Note : Les descriptions sont présentées dans la langue officielle dans laquelle elles ont été soumises.
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OUTER MEMBRANE PROTEIN A. PEPTIDOGLYCAN-ASSOCIATED
LIPOPROTEIN, AND MUREIN LIPOPROTEIN AS THERAPEUTIC TARGETS
FOR TREATMENT OF SEPSIS
Related Application
This application claims priority to U.S. Provisional Patent Application No.
60/149,960, filed August 20, 1999, the entire contents of which is hereby
incorporated by
reference.
Field Of Invention
The present invention relates to pharmaceutical compositions and methods
useful for
preventing and treating Gram-negative sepsis. In particular, the invention
arises from the
identification of three outer membrane proteins conserved among a number of
Gram-negative
bacteria and relates to antibodies directed to them.
Background Of Invention
Infections due to Gram-negative bacteria are a major cause of morbidity and
mortality. Gram-negative sepsis, the systemic inflammatory response to the
microbial
invasion, often first manifested as fever, hypothermia, tachycardia, or
tachypnea, can
progress to life-threatening hypotension and organ failure. While microbial
invasion of the
bloodstream is common in advanced stages of sepsis, localized Gram-negative
infections can
lead to Gram-negative sepsis on the basis of host responses to local or
systemic release of
microbial signals. Such microbial signals frequently arise from bacterial cell
wall
components such as lipopolysaccharide (LPS), also known as endotoxin.
The notion of treating Gram-negative sepsis with antibody directed to
conserved cell
wall components is supported by many studies over the last thirty years that
show that
administration of polyclonal antisera raised to rough mutant bacteria protects
in Gram-
negative sepsis caused by heterologous Gram-negative bacteria. Chedid L et
al., A proposed
mechanism for natural immunity to enterobacterial pathogens, Jlmmunol 100:292-
301
(1968); Braude AI et al., Treatment and prevention of intravascular
coagulation with
antiserum to endotoxin, Jlnfect Dis 128:5157-5164 (1973); McCabe WR et al.,
Cross-
reactive antigens: Their potential for immunization-induced immunity to gram-
negative
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bacteria, Jlnfect Dis 136:S161-5166 (1977); McCabe WR et al., Immunization
with rough
mutants of Salmonella minnesota: protective activity of IgM and IgG antibody
to the 8595
(Re Chemotype) mutant, Jlnfect Dis 158:291-300 (1988); Ziegler EJ et al.,
Treatment of
gram-negative bacteremia and shock with human antiserum to a mutant
Escherichia coli, N
Engl JMed 307:1225-1230 (1982); Baumgartner J et al., Prevention of Gram-
negative shock
and death in surgical patients by antibody to endotoxin core glycolipid,
Lancet 59-63 (1985).
It has generally been assumed that immunoglobulins in antisera to rough mutant
strains such as Escherichia coli J5 and Salmonella minnesota Re595 protect by
binding to
conserved core components (lipid A and core oligosaccharide) of
lipopolysaccharide (LPS).
There has not, however, been direct evidence that anti-core monoclonal
antibodies protect,
with the exception of one monoclonal antibody, WN1 222-5, which has been
reported to bind
core structures of LPS from heterologous enteric Gram-negative bacteria and to
protect from
endotoxin challenge in rabbits and mice. Di Padova FE et al., A broadly cross-
protective
monoclonal antibody binding to Escherichia coli and Salmonella
lipopolysaccharides, Infect
Immun 61:3863-3872 (1993). In addition, it has been difficult to directly
demonstrate
substantial increased binding to LPS from heterologous Gram-negatives by the
immunoglobulins in polyclonal antiserum to E. coli J5. Siber GR et al., Cross-
reactivity of
rabbit antibodies to lipopolysaccharides of Escherichia coli J5 and other gram-
negative
bacteria, Jlnfect Dis 152:954-964 (1985); Warren HS et al., Endotoxin
neutralization with
rabbit antisera to Escherichia coli J5 and other gram-negative bacteria,
Infect Immun
55:1668-1673 (1987). Nonetheless, although antisera raised to heat-killed
rough strains have
been reported to protect, the exact mechanism by which this protection occurs
remains
elusive.
We previously reported that immunoglobulin G (IgG) in these antisera bind only
weakly to LPS from heterologous Gram-negative strains. Siber GR et al., Cross-
reactivity of
rabbit antibodies to lipopolysaccharides of Escherichia coli J5 and other gram-
negative
bacteria, Jlnfect Dis 152:954-964 (1985); Warren HS et al., Endotoxin
neutralization with
rabbit antisera to Escherichia coli J5 and other gram-negative bacteria,
Infect Immun
55:1668-1673 (1987). The resounding clinical failure of anti-lipid A
monoclonal antibodies
(that were based upon these antisera) has resulted in decreased interest in
this approach.
We also recently reported that IgG in polyclonal antiserum raised to heat-
killed E. coli
J5 bacteria (J5 antiserum) binds to three conserved Gram-negative bacterial
outer membrane
proteins (OMPs). These OMPs are exposed on the surface of bacteria incubated
in human
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serum and are released into human serum in complexes that also contain LPS.
Hellman J et
al., Antiserum against Escherichia coli JS contains antibodies reactive with
outer membrane
proteins of heterologous Gram-negative bacteria, Jlnfect Dis 176:1260-1268
(1997). The
identities of the antigens bound by JS antiserum are unknown.
Summary Of The Invention
The invention solves these and other problems by providing methods and
compositions for treating infection and sepsis due to Gram-negative bacteria.
In a first aspect the invention provides a vaccine composition comprising an
effective
amount of an isolated outer membrane protein (OMP) selected from the group
consisting of
outer membrane protein A (OmpA), peptidoglycan-associated lipoprotein (PAL),
murein
lipoprotein (MLP), and immunogenic portions thereof, in a pharmaceutically
suitable carrier.
The vaccine is believed to be useful for active immunization against multiple
Gram-negative
bacteria. The vaccine can include an adjuvant, which can preferably be
selected from
Al(OH)3, A1P04, QS21, CpG, and any combination of these.
In one embodiment the isolated OMP is OmpA. In another embodiment the isolated
OMP is PAL. In yet another embodiment the isolated OMP is MLP.
In another aspect the invention provides an adjuvant comprising an effective
amount
of an isolated OMP selected from the group consisting of OmpA, PAL, MLP, and
any
combination thereof, in a pharmaceutically acceptable carrier. The adjuvant
can be used in
association with exposure to an antigen other than OmpA, PAL, MLP, and
immunogenic
portions thereof.
The invention in another aspect provides a pharmaceutical composition
comprising an
effective amount of an isolated polypeptide that binds specifically to at
least a portion of
OmpA, PAL, or MLP, in a pharmaceutically suitable Garner. In various
embodiments the
isolated polypeptide can include a monoclonal antibody, a derivative of a
monoclonal
antibody, a polyclonal antibody, or a synthetic polypeptide. The antibody can
be a human
antibody or a humanized antibody. Preferably the antibody or antibody
derivative is a human
antibody. The polyclonal antibody is distinct from polyclonal antibody raised
against killed
whole Gram-negative bacteria and unfractionated cell walls from Gram-negative
bacteria.
Preferably the synthetic polypeptide is a member of a combinatorial library of
synthetic
polypeptides.
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In yet another aspect the invention provides an immortal cell line that
secretes a
polypeptide that binds specifically to an outer membrane protein selected from
the group
consisting of OmpA, PAL, MLP, and any immunogenic portion thereof. In certain
embodiments the secreted polypeptide is a monoclonal antibody. In other
embodiments the
secreted polypeptide includes a fragment of a monoclonal antibody. In
preferred
embodiments the monoclonal antibody or fragment of a monoclonal antibody is of
human
origin. In alternative preferred embodiments the monoclonal antibody or
fragment of a
monoclonal antibody is humanized.
In one embodiment of this aspect of the invention the isolated OMP is OmpA. In
another embodiment the isolated OMP is PAL. In yet another embodiment the
isolated OMP
is MLP.
Another aspect of the invention is a method of immunizing a subject against
infection
due to Gram-negative bacteria wherein a subject is administered an isolated
outer membrane
protein antigen selected from the group consisting of OmpA, PAL, MLP, and any
immunogenic portion thereof, in a pharmaceutically suitable carrier, in an
amount effective
for inducing protection against infection due to Gram-negative bacteria.. In
one embodiment
of this aspect of the invention the isolated OMP is OmpA. In another
embodiment the
isolated OMP is PAL. In yet another embodiment the isolated OMP is MLP. In a
further
embodiment the methods of active vaccination can include administration of an
adjuvant.
Preferably the adjuvant is selected from Al(OH)3, A1P04, QS21, CpG, and any
combination
of these. In certain embodiments the antigen is administered subcutaneously.
In alternative
embodiments, the antigen is administered intradermally, intramuscularly, or
mucosally.
In another aspect the invention provides a method of treating a subject
infected with
Gram-negative bacteria, wherein the method involves administering to a subject
who has an
infection with Gram-negative bacteria an isolated polypeptide that binds
specifically to at
least a portion of an outer membrane protein selected from the group
consisting of OmpA,
PAL, and MLP, in an amount effective to treat the infection. In a preferred
embodiment the
amount is effective to inhibit Gram-negative sepsis. In another preferred
embodiment the
amount is effective to inhibit growth of the Gram-negative bacteria in vivo.
In various embodiments of this aspect of the invention, the isolated
polypeptide can
be a monoclonal antibody, a derivative of a monoclonal antibody, a polyclonal
antibody, or a
member of a library of synthetic polypeptides.
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In certain embodiments the administered amount of polypeptide is effective to
enhance clearance of Gram-negative bacteria from blood of the subject. In
other
embodiments the administered amount of polypeptide is effective to enhance
clearance of
insoluble fragments of Gram-negative bacteria from blood of the subject.
In yet other embodiments the administered amount of polypeptide is effective
to
neutralize Gram-negative bacteria in blood of the subj ect. In other
embodiments the
administered amount of polypeptide is effective to neutralize insoluble
fragments of Gram-
negative bacteria in blood of the subject.
According to another embodiment the administered amount of polypeptide is
effective
to opsonize Gram-negative bacteria in blood of the subject. In a further
embodiment the
administered amount of polypeptide is effective to opsonize insoluble
fragments of Gram-
negative bacteria in blood of the subject.
In certain embodiments the method also involves administration of an effective
amount of an immune system stimulant. In preferred embodiments the immune
system
stimulant is a cytokine. In other preferred embodiments the immune system
stimulant is an
adjuvant.
In a further aspect the invention provides a method of treating a subject with
Gram-
negative sepsis, wherein a subject in need of such treatment is administered a
composition
containing an isolated polypeptide that binds specifically to at least a
portion of an outer
membrane protein selected from the group consisting of OmpA, PAL, and MLP, in
an
amount effective to inhibit sepsis-related release of at least one soluble
factor into blood or
tissue of the subject.
In certain embodiments of this aspect of the invention, the soluble factor is
released
by Gram-negative bacteria upon their exposure to serum. In one embodiment the
soluble
factor is LPS. In another embodiment the soluble factor is OmpA. In a further
embodiment
the soluble factor is PAL. In yet another embodiment the soluble factor is
MLP.
In certain other embodiments of this aspect of the invention, the soluble
factor is
released by cells of the infected host. In some embodiments the soluble factor
is a cytokine.
In yet other embodiments the released factor is selected from IL-1, IL-6, TNF-
a, high
mobility group-1 protein (HMG-1), migration inhibitory factor (MIF),
chemokines, and nitric
oxide.
In yet a further aspect the invention provides a method of treating a subject
who has
Gram-negative sepsis, involving administering to a subject in need of such
treatment a
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composition comprising an isolated polypeptide that binds specifically to at
least a portion of
an outer membrane protein selected from the group consisting of OmpA, PAL, and
MLP, in
an amount effective to enhance clearance of at least one sepsis-related
soluble factor released
by Gram-negative bacteria into blood of the subject.
In one embodiment the soluble factor is LPS. In another embodiment the soluble
factor is OmpA. In a further embodiment the soluble factor is PAL. In yet
another
embodiment the soluble factor is MLP.
The invention will be more fully understood by reference to the following
figures and
detailed description.
Brief Description Of The Fi ures
Figure 1 depicts an immunoblot (Milliblot) analysis of monoclonal antibodies
using
lysates of mid-log phase E. coli 06 bacteria as antigen. Primary antibodies
for the
immunoblots include polyclonal mouse anti-JS IgG (lane 1 ) and monoclonal
antibodies 2D3
(lane 2), 6D7 (lane 3), and 1C7 (lane 4). Estimated molecular weights of the
bands (kDa) are
indicated at the left.
Figure 2 depicts an immunoblot analysis of OmpA-deficient bacteria. Mid-log
phase
bacteria are electrophoresed on 16% SDS-polyacrylamide gels and transferred to
nitrocellulose. Staining antibodies include polyclonal rabbit anti-JS IgG
(left panel), and a
monoclonal antibody directed to the 35 kDa OMP (2D3, right panel). Bacterial
strains are:
wild type OmpA+E. coli 018:K1:H7 (lane 1); E91, an OmpA-deleted mutant of E.
coli
018:K1:H7 (lane 2); E69, and an OmpA-restored mutant of E. coli 018:K1:H7
(lane 3).
Molecular weight markers (kDa) are as at the left.
Figure 3 depicts an immunoblot analysis of recombinant OmpA. Primary
antibodies
include polyclonal mouse anti-JS IgG (left panel) and the monoclonal antibody
directed
against the 35 kDa OMP (2D3, right panel).
Figure 4 depicts an immunoblot analysis of PAL-deficient bacteria. Staining
antibodies include polyclonal rabbit anti-JS IgG (left panel), and monoclonal
antibody 6D7
(right panel). Bacterial strains are: E coli K12 p400 containing PAL (lane 1);
CH202, a
PAL-deficient mutant of E. coli K12 p400 (lane 2); CH202 prC2, a PAL-restored
mutant of
CH202 (lane 3); E. coli K12 1292 containing PAL (lane 4); JC7752, a PAL-
deficient mutant
of 1292 (lane 5); and JC7752 p417, a PAL-restored mutant of JC7752 (lane 6).
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WO 01/13948 PCT/US00/22736
Figure 5 depicts an immunoblot analysis of MLP-deficient bacteria. Staining
antibodies include polyclonal rabbit anti-JS IgG (left panel), and monoclonal
antibody 1 C7
(right panel). Bacterial strains are: E. coli 018K+ (lane 1); E. coli K12
JE5505, an MLP-
deficient mutant of E. coli K12 (lane 2); and E. coli K12AT1360, a closely
related isolate of
E. coli K12 containing MLP (lane 3).
Figure 6 depicts an immunoblot analysis of OMP-containing samples released
into
human serum. Eluted samples were stained with marine monoclonal IgGs directed
against
OmpA (2D3), PAL (6D7), and MLP (1C7) (left three panels), and with polyclonal
mouse
anti-JS IgG and a marine monoclonal IgG directed to the O-polysaccharide chain
of E. coli
018 LPS (right two panels). Samples for each panel were affinity-purified
with: rabbit anti-
JS IgG (lane 1), rabbit O-chain specific anti-LPS IgG (lane 2), and normal
rabbit IgG (lane
3). Molecular weight markers are as indicated.
Figure 7 depicts an immunoblot analysis of bacterial fragments released into
the
blood of burned rats with E coli 018K+ sepsis. Blots obtained from two
representative rats
are shown. Lanes correspond to samples from affinity purified plasma collected
from
bacteremic rats prior to (lane 1) and 3 hours after (lanes 2, 3, 4)
intravenous administration of
ceftazidime. Antigens were eluted from polyclonal rabbit anti-JS IgG (lanes l
and 2), normal
rabbit IgG (lane 3), and polyclonal rabbit IgG directed against the O-
polysaccharide side
chain of E. coli 018 LPS (lane 4) and developed with a mixture of monoclonal
antibodies
directed against each of the three OMPs (2D3, 6D7, and 1C7). Black arrows to
the right of
the blots indicate the 5-9 kDa, 18 kDa, and 35 kDa OMPs. White arrows to right
of the
figure indicate cross-reactive IgG bands (amplified by the more sensitive
chemiluminescence
technique). Molecular weight markers (kDa) are at the left.
Detailed Description
The present invention relates to three outer membrane proteins released by
Gram-
negative bacteria when the latter are incubated in human serum. The same outer
membrane
proteins are released into the circulation in an experimental model of sepsis,
and they are
bound by IgG in the cross-protective antiserum raised to Escherichia coli JS
(JS antiserum).
It has now been discovered that the identities of the three outer membrane
proteins are outer
membrane protein A (OmpA), peptidoglycan-associated lipoprotein (PAL), and
murein
lipoprotein (MLP)
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_g_
OmpA was initially described by Henning and coworkers in 1975. Hindennach I
and
Henning U, Eur JBiochem 59:207-213(1975); Garten W et al., Eur JBiochem 59:215-
221
(1975). It has 325 amino acid residues and exhibits heat-modifiable
electrophoretic mobility
on SDS-PAGE. Chen R et al., Proc Natl Acad Sci USA 77:4592-4596 (1980);
Nakamura K
and Mizushima S, JBiochem 80:1411-1422 (1976). The N-terminal domain of OmpA
is
comprised of 177 amino acids and is believed to traverse the outer membrane
eight times.
Klose M et al., JBiol Chem 268:25664-25670 (1993). The C-terminal domain is
believed to
protrude into the periplasmic space. OmpA is involved in maintaining the shape
of bacteria,
serves as a phage receptor and a receptor for F-mediated conjugation, and has
limited pore-
forming properties. Sonntag I et al., JBacteriol 136:280-285 (1978); Sugawara
E and
Nikaido H, JBiol Chem 267:2507-2511 (1992); Sugawara E and Nikaido H, JBiol
Chem
269:17981-17987 (1994). OmpA enhances uptake of LPS into macrophages and has
been
reported to be involved in E. coli invasion of the central nervous system.
Korn A et al., Infect
Immure 63:2697-2705 (1995); Prasadarao NV et al., Infect Immure 64:146-153
(1996). An
OmpA-deficient mutant of the virulent bacterial strain, E coli 018K1 was shown
to be less
virulent that its OmpA+ parent strain in neonatal rat and embryonated chick
egg models of
sepsis. Weiser JN and Gotschlich EC, Infect Immure 59:2252-2258 (1991).
PAL was initially characterized and described by Mizuno. Mizuno T, JBiochem
89:1039-1049 (1981). It has 173 amino acid residues and is closely, but not
covalently,
associated with the peptidoglycan layer. Lazzaroni J-C and Portalier R, Mol
Microbiol
6:735-742 (1992); Mizuno T, JBiochem 89:1039-1049 (1981); Mizuno T, JBiochem
86:991-
1000 (1979). PAL has a hydrophobic region of 22 amino acids at the N-terminal
domain that
interacts with the outer membrane. Lazzaroni J-C and Portalier R, Mol
Microbiol 6:735-742
(1992). The C-terminal domain is involved in interactions with the
peptidoglycan layer.
Lazzaroni J-C and Portalier R, Mol Microbiol 6:735-742 (1992).
MLP was first described and characterized by Braun. Hantke K and Braun V, Eur
J
Biochem 34:284-296 (1973); Braun V and Wolff H, Eur JBiochem 14:387-391
(1970);
Braun V and Bosch V, Eur JBiochem 28:51-69 (1972). It is the most abundant
outer
membrane protein. Braun V and Wolff H, Eur JBiochem 14:387-391 (1970). MLP has
58
amino acid residues and exists in two forms, a free form and a form that is
covalently linked
to peptidoglycan by the C-terminal domain. Braun V and Bosch V, Eur JBiochem
28:51-69
(1972); Braun V, Biochim Biophys Acta 415:335-377 (1975). Recently Zhang
reported that
MLP induces lethal shock in a strain of mouse (C3H/HeJ) that is genetically
hyporesponsive
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to LPS. Zhang H et al., Jlmmunol 159:4868-4878 (1997). Furthermore, they found
that
MLP was synergistic with LPS for lethal toxicity.
Applicants previously have shown that epitopes of three proteins are exposed
on the
surface of bacteria that have been incubated in human serum, and that
antiserum raised to a
rough mutant vaccine of E. coli JS results in high titers of antibodies that
bind to the same
three proteins on the bacterial surface. The identity of two of these proteins
as PAL and MLP
is surprising, as both proteins are situated in the deep periplasmic space and
only short N-
terminal segments are believed to interact with the outer membrane. Lazzaroni
J-C and
Portalier R, Mol Microbiol 6:735-742 (1992); Steinemann S et al., Arterioscler
Thromb
14:1202-1209 (1994). Therefore, the increased clearance of heterologous smooth
bacterial
strains by infusion of antiserum to E. coli JS (Sakulramrung R and Dominigue
G.J, Jlnfect
Dis 151:995-1004 (1985)) may be mediated by binding of immunoglobulin in this
antiserum
to epitopes of OmpA, PAL, and MLP on the bacterial surface.
Circulating bacterial toxins are believed to be important in the pathogenesis
of Gram-
negative sepsis, but little is actually known about the composition of
released bacterial
components. Most studies have focused on release of LPS, and it has been
assumed that LPS
is released in membrane blebs that then disaggregate into LPS monomers. Tesh
VL et al., J
Immunol 137:1329-1335 (1986); Tesh VL and Morrison DC, Jlmmunol 141:3523-3531
(1988); Danner RL et al., Chest 99:169-175 (1991); Pearson FC et al., JClin
Microbiol
21:865-868 (1985); Winchurch RA et al., Surgery 102:808-812 (1987); Wessels BC
et al.,
Crit Care Med 16:601-605 (1988); Brandtzaeg P et al., Regul Pept 24:37-44
(1989); van
Deventer SJ et al., Lancet 1:605-609 (1988); Natanson C et al., JClin Invest
83:243-251
(1989); Shenep JL et al., Jlnfect Dis 157:565-568 (1988); Munford RS et al.,
JClin Invest
70:877-888 (1982). Prior studies have shown that live bacteria incubated in
human serum
release fragments containing OMPs and LPS (OMP/LPS complexes) that can be
affinity-
purified using antibodies directed to the O-polysaccharide side chain of LPS.
Hellman J et
al., Jlnfect Dis 176:1260-1268 (1997); Freudenberg MA et al., Microb Pathog
10:93-104
(1991). Freudenberg reported that samples that were affinity-purified from
filtrates of serum-
exposed Salmonella abortus equi bacteria using anti-LPS IgG also contained
OmpA and a
second protein of MW 17 kDa that was not identified. Freudenberg MA et al.,
Microb
Pathog 10:93-104 (1991).
Applicants now have found that OMP/LPS complexes that contain at least three
OMPs are released in vivo into the bloodstream in an infected burn model of
Gram-negative
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sepsis. The 18 kDa OMP is also released into septic rat blood in a form that
is separate from
the OMP/LPS complexes and is selectively affinity purified by IgG in antiserum
raised to
heat-killed E. coli JS bacteria.
Although many studies report that proteins that are tightly associated with
LPS are
biologically active, the role of OMPs in the pathogenesis of sepsis has not
been defined.
Melchers F et al., JExp Med 142:473-482 (1975); Doe WF et al., JExp Med
148:557-568
(1978); Goodman GW and Sultzer BM, Jlmmunol 122:1329-1334 (1979); Goodman GW
and Sultzer BM, Infect Immun 24:685-696 (1979); Chen Y et al., Infect Immun
28:178-184
(1980); Goldman RC et al., Jlmmunol 127:1290-1294 (1981); Galdiero F et al.,
Infect Immun
46:559-563 (1984); Bjornson BH et al., Infect Immun 56:1602-1607 (1988); Johns
MA et al.,
Infectlmmun 56:1593-1601 (1988); Hauschildt S et al., EurJImmunol20:63-68
(1990);
Porat R et al., Infect Immun 60:1756-1760 (1992); Mangan DF et al., Infect
Immun 60:1684-
1686 (1992); Galdiero F et al., Infect Immun 61:155-161 (1993); Manthey CL et
al., J
Immunol 153:2653-2663 (1994); Snapper CM et al., Jlmmunol 155:5582-5589
(1995); Korn
A et al., Infect Immun 63:2697-2705 (1995); Zhang H et al., Jlmmunol 159:4868-
4878
(1997); Giambartolomei GH et al., Infect Immun 67:140-147 (1999). Given these
studies and
the previously described protective efficacy of JS antiserum, it appears that
OMPs play a role
in the pathogenesis of Gram-negative sepsis.
The invention in one aspect provides vaccine compositions that incorporate an
effective amount of at least one isolated outer membrane protein selected from
OmpA, PAL,
MLP, and any immunogenic portion thereof, prepared in a pharmaceutically
suitable carrier.
In a related aspect, the invention provides a method of making a vaccine
composition,
involving placing an effective amount of at least one isolated outer membrane
protein
selected from OmpA, PAL, MLP, and any immunogenic portion thereof, in a
pharmaceutically suitable Garner.
The term "effective amount" as used herein refers to the amount necessary or
sufficient to realize a desired biologic effect. For example, an effective
amount of an isolated
outer membrane protein in a vaccine composition is that amount necessary to
cause the
development of an antigen-specific immune response upon exposure to the OMP,
thus
inducing protection. The effective amount for any particular application can
vary depending
on such factors as the particular OMP being administered, the particular
adjuvant (if any)
used in conjunction with the antigen, the route of administration, the size of
the subject, the
competence of the immune system of the subject, or the severity of the disease
or condition.
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One of ordinary skill in the art can empirically determine the effective
amount of a particular
OMP antigen without necessitating undue experimentation.
The formulations of the invention are administered in pharmaceutically
acceptable
solutions, which may routinely contain pharmaceutically acceptable
concentrations of salt,
buffering agents, preservatives, compatible carriers, adjuvants, and
optionally other
therapeutic ingredients.
For use in therapy, an effective amount of the vaccine composition or
pharmaceutical
composition can be administered to a subject by any mode allowing the OMP
antigen to be
taken up by the appropriate target cells. "Administering" the vaccine or
pharmaceutical
composition of the present invention may be accomplished by any means known to
the
skilled artisan. Preferred routes of administration include but are not
limited to oral,
transdermal (e.g. via a patch), parenteral injection (subcutaneous,
intradermal, intravenous,
intramuscular, intraperitoneal, intrathecal, etc.), or mucosal (intranasal,
intratracheal,
inhalation, and intrarectal, intravaginal etc). An injection may be in a bolus
or a continuous
infusion.
For example the vaccine and pharmaceutical compositions according to the
invention
are often administered by intramuscular or intradermal injection, or other
parenteral means,
or by biolistic "gene-gun"application to the epidermis. They may also be
administered by
intranasal application, inhalation, topically, intravenously, orally, or as
implants, and even
rectal or vaginal use is possible. Suitable liquid or solid pharmaceutical
preparation forms
are, for example, aqueous or saline solutions for injection or inhalation,
microencapsulated,
encochleated, coated onto microscopic gold particles, contained in liposomes,
nebulized,
aerosols, pellets for implantation into the skin, or dried onto a sharp object
to be scratched
into the skin. The pharmaceutical compositions also can include granules,
powders, tablets,
coated tablets, (micro)capsules, suppositories, syrups, emulsions,
suspensions, creams, drops
or preparations with protracted release of active compounds, in whose
preparation excipients
and additives and/or auxiliaries such as disintegrants, binders, coating
agents, swelling
agents, lubricants, flavorings, sweeteners or solubilizers are customarily
used as described
above. The pharmaceutical compositions are suitable for use in a variety of
drug delivery
systems. For a brief review of present methods for drug delivery, see Langer,
Science
249:1527-1533 (1990), which is incorporated herein by reference.
The pharmaceutical compositions are preferably prepared and administered in
dose
units. Liquid dose units are vials or ampoules for injection or other
parenteral administration.
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Solid dose units are tablets, capsules and suppositories. For treatment of a
patient, depending
on activity of the compound, manner of administration, purpose of the
immunization (i.e.,
prophylactic or therapeutic), nature and severity of the disorder, age and
body weight of the
patient, different doses may be necessary. The administration of a given dose
can be carried
out both by single administration in the form of an individual dose unit or
else several smaller
dose units. Multiple administration of doses at specific intervals of weeks or
months apart is
usual for boosting the antigen-specific responses.
The antigens and adjuvants may be administered per se (neat) or in the form of
a
pharmaceutically acceptable salt. When used in medicine the salts should be
pharmaceutically acceptable, but non-pharmaceutically acceptable salts may
conveniently be
used to prepare pharmaceutically acceptable salts thereof. Such salts include,
but are not
limited to, those prepared from the following acids: hydrochloric,
hydrobromic, sulphuric,
nitric, phosphoric, malefic, acetic, salicylic, p-toluene sulphonic, tartaric,
citric, methane
sulphonic, formic, malonic, succinic, naphthalene-2-sulphonic, and benzene
sulphonic. Also,
such salts can be prepared as alkaline metal or alkaline earth salts, such as
sodium, potassium
or calcium salts of the carboxylic acid group.
Suitable buffering agents include: acetic acid and a salt (1-2% w/v); citric
acid and a
salt (1-3% w/v); boric acid and a salt (0.5-2.5% w/v); and phosphoric acid and
a salt (0.8-2%
w/v). Suitable preservatives include benzalkonium chloride (0.003-0.03% w/v);
chlorobutanol (0.3-0.9% w/v); parabens (0.01-0.25% w/v) and thimerosal (0.004-
0.02% w/v).
The pharmaceutical compositions of the invention contain an effective amount
of an
antigen optionally included in a pharmaceutically suitable carrier. The term
"pharmaceutically suitable carrier" means one or more compatible solid or
liquid filler,
diluants or encapsulating substances which are suitable for administration to
a human or other
vertebrate animal. The term "carrier" denotes an organic or inorganic
ingredient, natural or
synthetic, with which the active ingredient is combined to facilitate the
application. The
components of the pharmaceutical compositions also are capable of being
comingled with the
compounds of the present invention, and with each other, in a manner such that
there is no
interaction which would substantially impair the desired pharmaceutical
efficiency.
Compositions suitable for parenteral administration conveniently comprise
sterile
aqueous preparations, which can be isotonic with the blood of the recipient.
Among the
acceptable vehicles and solvents are water, Ringer's solution, phosphate
buffered saline, and
isotonic sodium chloride solution. In addition, sterile, fixed oils are
conventionally employed
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as a solvent or suspending medium. For this purpose any bland fixed mineral or
non-mineral
oil may be employed including synthetic mono- or di-glycerides. In addition,
fatty acids such
as oleic acid find use in the preparation of injectables. Carrier formulations
suitable for
subcutaneous, intramuscular, intraperitoneal, intravenous, etc.
administrations may be found
in Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.
The compositions may conveniently be presented in unit dosage form and may be
prepared by any of the methods well known in the art of pharmacy. All methods
include the
step of bringing the compounds into association with a carrier which
constitutes one or more
accessory ingredients. In general, the compositions are prepared by uniformly
and intimately
bringing the compounds into association with a liquid carrier, a finely
divided solid carrier, or
both, and then, if necessary, shaping the product.
Other delivery systems can include time-release, delayed release or sustained
release
delivery systems. Such systems can avoid repeated administrations of the
compounds,
increasing convenience to the subject and the physician. Many types of release
delivery
systems are available and known to those of ordinary skill in the art. They
include polymer-
based systems such as poly(lactide-glycolide), copolyoxalates,
polycaprolactones,
polyesteramides, polyorthoesters, polyhydroxybutyric acid, and polyanhydrides.
Microcapsules of the foregoing polymers containing drugs are described in, for
example, U.S.
Patent 5,075,109. Delivery systems also include non-polymer systems that are:
lipids
including sterols such as cholesterol, cholesterol esters and fatty acids or
neutral fats such as
mono-, di-, and tri-glycerides; hydrogel release systems; sylastic systems;
peptide based
systems; wax coatings; compressed tablets using conventional binders and
excipients;
partially fused implants; and the like. Specific examples include, but are not
limited to: (a)
erosional systems in which an agent of the invention is contained in a form
within a matrix
such as those described in U.S. Patent Nos. 4,452,775, 4,675,189, and
5,736,152, and (b)
diffusional systems in which an active component permeates at a controlled
rate from a
polymer such as described in U.S. Patent Nos. 3,854,480, 5,133,974 and
5,407,686. In
addition, pump-based hardware delivery systems can be used, some of which are
adapted for
implantation.
As used herein in reference to an OMP or other polypeptide, the term
"isolated"
means separated from its native environment in sufficiently pure form so that
it can be
manipulated or used for any one of the purposes of the invention. An isolated
compound
refers to a compound which represents at least 10 percent of the compound
present in the
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mixture and exhibits a detectable (i.e., statistically significant) biological
activity when tested
in conventional biological assays such as those described herein. Preferably
the isolated
compound represents at least 50 percent of the mixture; more preferably at
least 80 percent of
the mixture; and most preferably at least 90 percent or at least 95 percent of
the mixture.
Thus, isolated means sufficiently pure to be used (i) to raise and/or isolate
antibodies, (ii) as a
reagent in an assay, or (iii) for sequencing, etc.
Thus, in the preferred embodiments, the isolated outer membrane proteins are
immunogenic and can be used to generate binding polypeptides (e.g.,
antibodies) for use in
diagnostic and therapeutic applications. Such binding polypeptides also are
useful for
detecting the presence, absence, and/or amounts of particular OMPs in a sample
such as a
biological fluid or biopsy sample.
The invention also provides isolated OMPs (including whole proteins and
partial
proteins), encoded by previously known nucleic acids. Outer membrane proteins
can be
isolated from biological samples including tissue or cell homogenates, and can
also be
expressed recombinantly in a variety of prokaryotic and eukaryotic expression
systems by
constructing an expression vector appropriate to the expression system,
introducing the
expression vector into the expression system, and isolating the recombinantly
expressed
protein. Short polypeptides, including antigenic peptides (such as are
presented by MHC
molecules on the surface of a cell for immune recognition) also can be
synthesized
chemically using well-established methods of peptide synthesis.
The term "outer membrane protein" as used herein in reference to the three
specific
OMPs OmpA, PAL, and MLP shall include both the polypeptide component alone and
the
polypeptide component in association with lipid. In this way, the term "outer
membrane
protein" can encompass the fact that PAL and MLP occur naturally as
lipoproteins. The
association between the polypeptide component and lipid can be covalent or non-
covalent.
The term "OmpA" as used herein refers to any of a number of immunologically
cross-
reactive cell wall polypeptide components from heterologous Gram-negative
bacteria known
in the art as outer membrane protein A or OmpA. As used herein, OmpA is
distinct from
LPS and exemplified by, but not limited to, OmpA of E coli K12, GenBank
accession no.
P02934. It is recognized that OmpA can be released into human serum in vitro
and in vivo in
complexes that also contain LPS. The term "OmpA" as used herein shall include
both the
polypeptide component alone and the polypeptide component in association with
lipid.
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The term "PAL" as used herein refers to any of a number of immunologically
cross-
reactive lipoprotein cell wall components from heterologous Gram-negative
bacteria known
in the art as peptidoglycan-associated lipoprotein or PAL. As used herein, PAL
is distinct
from LPS and exemplified by, but not limited to, PAL of E coli K12, GenBank
accession no.
P07176. It is recognized that PAL can be released into human serum in vitro
and in vivo in
complexes that also contain LPS. The term "PAL" as used shall include both the
polypeptide
component alone and the polypeptide component in association with lipid.
The term "MLP" as used herein refers to any of a number of immunologically
cross-
reactive lipoprotein cell wall components from heterologous Gram-negative
bacteria known
in the art simply as lipoprotein, or as Braun's lipoprotein, murein
lipoprotein, or MLP. As
used herein, MLP is distinct from LPS and exemplified by, but not limited to,
MLP of E. coli
K12, GenBank accession no. P02937. It is recognized that MLP can be released
into human
serum in vitro and in vivo in complexes that also contain LPS. The term "MLP"
as used shall
include both the polypeptide component alone and the polypeptide component in
association
with lipid.
An "immunogenic portion" as used herein refers to any fragment of an isolated
OMP
that can, under appropriate conditions, induce an immune response. For an
immune response
involving antibodies, an immunogenic portion will include an antigenic
determinant which is
the target of antibody binding. With respect to proteins and polypeptides,
antigenic
determinants involve specific amino acid residues in a particular three-
dimensional
conformation. These amino acid residues must be exposed on the surface of the
protein or
polypeptide in order to be immunogenic. For an immune response involving T
cells, an
immunogenic portion of a protein or polypeptide is most often an
immunodominant
determinant or, alternatively, a cryptic determinant. Sercarz EE et al., Annu
Rev Immunol
11:729-766 (1993). T-cell response to both these types of determinants involve
antigen
processing, i.e., intracellular partial degradation of protein or polypeptide
into short
oligopeptides which are subsequently associated with major histocompatibility
complex
(MHC) molecules and presented on the surface of the T cell.
An "adjuvant" is any molecule or compound which can stimulate or augment the
stimulation of a humoral and/or cellular immune response. An adjuvant
typically is
administered in association with exposure to an antigen to enhance the immune
response to
the antigen. An immune system stimulant exerts a mitogenic effect on immune
system cells
and can cause increased cytokine expression by vertebrate lymphocytes. A
number of
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adjuvants are well known in the art. These can include, for instance,
adjuvants that create a
depot effect, immune-stimulating adjuvants, adjuvants that create a depot
effect and stimulate
the immune system, and mucosal adjuvants.
An adjuvant that creates a depot effect is an adjuvant that causes an antigen
to be
slowly released in the body, thus prolonging the exposure of immune cells to
the antigen.
This class of adjuvants includes but is not limited to alum (e.g., aluminum
hydroxide,
aluminum phosphate); or emulsion-based formulations including mineral oil, non-
mineral oil,
water-in-oil or oil-in-water-in oil emulsion, oil-in-water emulsions such as
Seppic ISA series
of Montanide adjuvants (e.g., Montanide ISA 720, AirLiquide, Paris, France);
MF-59 (a
squalene-in-water emulsion stabilized with Span 85 and Tween 80; Chiron
Corporation,
Emeryville, CA; and PROVAX (an oil-in-water emulsion containing a stabilizing
detergent
and a micelle-forming agent; IDEC, Pharmaceuticals Corporation, San Diego,
CA).
An immune-stimulating adjuvant is an adjuvant that causes direct activation of
a cell
of the immune system. It may, for instance, cause an immune cell to produce
and secrete
cytokines. This class of adjuvants includes but is not limited to saponins
purified from the
bark of the Q. saponaria tree, such as QS21 (a glycolipid that elutes in the
21 St peak with
HPLC fractionation; Aquila Biopharmaceuticals, Inc., Worcester, MA);
poly[di(carboxylatophenoxy)phosphazene (PCPP polymer; Virus Research
Institute, USA);
derivatives of lipopolysaccharides such as monophosphoryl lipid A (MPL; Ribi
ImmunoChem Research, Inc., Hamilton, MT), muramyl dipeptide (MDP; Ribi)
andthreonyl-
muramyl dipeptide (t-MDP; Ribi); OM-174 (a glucosamine disaccharide related to
lipid A;
OM Pharma SA, Meyrin, Switzerland); Leishmania elongation factor (a purified
Leishmania
protein; Corixa Corporation, Seattle, WA); and CpG DNA (WO 96/02555).
Adjuvants that create a depot effect and stimulate the immune system are those
compounds which have both of the above-identified functions. This class of
adjuvants
includes but is not limited to ISCOMS (immunostimulating complexes which
contain mixed
saponins, lipids and form virus-sized particles with pores that can hold
antigen; CSL,
Melbourne, Australia); SB-AS2 (SmithKline Beecham adjuvant system #2 which is
an oil-in-
water emulsion containing MPL and QS21; SmithKline Beecham Biologicals [SBB],
Rixensart, Belgium); SB-AS4 (SmithKline Beecham adjuvant system #4 which
contains
alum and MPL; SBB, Belgium); non-ionic block copolymers that form micelles
such as CRL
1005 (which contains a linear chain of hydrophobic polyoxpropylene flanked by
chains of
polyoxyethylene; Vaxcel, Inc., Norcross, GA); and Syntex Adjuvant Formulation
(SAF, an
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oil-in-water emulsion containing Tween 80 and a nonionic block copolymer;
Syntex
Chemicals, Inc., Boulder, CO).
A mucosal adjuvant is an that is capable of inducing a mucosal immune response
in a
subject when administered to a mucosal surface in conjunction with an antigen.
Mucosal
adjuvants include but are not limited to bacterial toxins: e.g., Cholera toxin
(CT); CT
derivatives including but not limited to CT B subunit (CTB) (Wu et al., 1998,
Tochikubo et
al., 1998); CTD53 (Val to Asp) (Fontana et al., 1995); CTK97 (Val to Lys)
(Fontana et al.,
1995); CTK104 (Tyr to Lys) (Fontana et al., 1995); CTD53/K63 (Val to Asp, Ser
to Lys)
(Fontana et al., 1995); CTH54 (Arg to His) (Fontana et al., 1995); CTN107 (His
to Asn)
(Fontana et al., 1995); CTEI 14 (Ser to Glu) (Fontana et al., 1995); CTE112K
(Glu to Lys)
(Yamamoto et al., 1997a); CTS61F (Ser to Phe) (Yamamoto et al., 1997a, 1997b);
CTS106
(Pro to Lys) (Douce et al., 1997, Fontana et al., 1995); and CTK63 (Ser to
Lys) (Douce et al.,
1997, Fontana et al., 1995); zonula occludens toxin (zot); Escherichia coli
heat-labile
enterotoxin (Labile Toxin, LT); LT derivatives including but not limited to LT
B subunit
(LTB) (Verweij et al., 1998); LT7K (Arg to Lys) (Komase et al., 1998; Douce et
al., 1995);
LT61F (Ser to Phe) (Komase et al., 1998); LT112K (Glu to Lys) (Komase et al.,
1998);
LT118E (Gly to Glu) (Komase et al., 1998); LT146E (Arg to Glu) (Komase et al.,
1998);
LT192G (Arg to Gly) (Komase et al., 1998); LTK63 (Ser to Lys) (Marchetti et
al., 1998;
Douce et al., 1997, 1998; Di Tommaso et al., 1996); and LTR72 (Ala to Arg)
(Giuliani et al.,
1998); Pertussis toxin (PT) (Lycke et al., 1992; Spangler BD, 1992; Freytag
and Clemments,
1999; Roberts et al., 1995; Wilson et al., 1995) including PT-9K/129G (Roberts
et al., 1995;
Cropley et al., 1995); toxin derivatives (Holmgren et al., 1993; Verweij et
al., 1998; Rappuoli
et al., 1995; Freytag and Clements, 1999); lipid A derivatives (e.g.,
monophosphoryl lipid A,
MPL) (Sasaki et al., 1998; Vancott et al., 1998); muramyl dipeptide (MDP)
derivatives
(Fukushima et al., 1996; Ogawa et al., 1989; Michalek et al., 1983; Morisaki
et al., 1983);
bacterial outer membrane proteins (e.g., outer surface protein A (OspA);
lipoprotein of
Borrelia burgdorferi; outer membrane protein of Neisseria meningitidis)
(Marinaro et al.,
1999; Van de Verg et al., 1996); oil-in-water emulsions (e.g., MF59)
(Barchfield et al., 1999;
Verschoor et al., 1999; O'Hagan, 1998); aluminum salts (Isaka et al., 1998,
1999); and
saponins (e.g., QS21) (Aquila Biopharmaceuticals, Inc., Worcester, MA) (Sasaki
et al., 1998;
MacNeal et al., 1998), ISCOMS; MF-59 (a squalene-in-water emulsion stabilized
with Span
85 and Tween 80; Chiron Corporation, Emeryville, CA); the Seppic ISA series of
Montanide
adjuvants (e.g., Montanide ISA 720; AirLiquide, Paris, France); PROVAX (an oil-
in-water
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emulsion containing a stabilizing detergent and a micelle-forming agent; IDEC
Pharmaceuticals Corporation, San Diego, CA); Syntext Adjuvant Formulation
(SAF; Syntex
Chemicals, Inc., Boulder, CO); poly[di(carboxylatophenoxy)phosphazene (PCPP
polymer;
Virus Research Institute, USA); and Leishmania elongation factor (Corixa
Corporation,
Seattle, WA).
The invention in another aspect provides an adjuvant that includes an
effective
amount of at least one isolated outer membrane protein selected from OmpA,
PAL, MLP, and
any combination thereof. It is believed that these compounds are useful as
adjuvants
themselves. It is well known in the art that various killed whole bacteria in
addition to killed
M. tuberculosis are useful as adjuvants. LPS itself is a powerful adjuvant,
but its utility is
severely restricted by its very significant toxicity. Since isolated outer
membrane proteins
appear to have biologic activity separate from LPS, and because LPS
preparations commonly
contain at least some outer membrane proteins, it is believed that these outer
membrane
proteins themselves have adjuvant activity.
In yet another aspect the invention provides pharmaceutical compositions
useful for
treating a subject infected with Gram-negative bacteria. Such pharmaceutical
compositions
include an isolated polypeptide that binds specifically to at least a portion
of OmpA, PAL, or
MLP, prepared in a pharmaceutically suitable carrier. The binding interaction
between the
pharmaceutical composition and the outer membrane protein will typically but
not
necessarily involve a non-covalent association between them. The effect of the
specific
binding in vivo can result in passive immunization. Mechanisms by which such
passive
immunization is believed to exert an effect are disclosed below. The effect of
the specific
binding in vivo and in vitro can also lead to functional deactivation of the
outer membrane
protein by, for example, sequestering or otherwise making inaccessible a
biologically active
site on the OMP. The types of pharmaceutical compositions contemplated in this
aspect of
the invention include monoclonal antibodies, fragments of monoclonal
antibodies, agents
formed in part by monoclonal antibodies or fragments thereof, polyclonal
antibodies, and
synthetic polypeptides that may be generated as part of a combinatorial
library of such
polypeptides.
In a related aspect, the invention further provides a method of making a
pharmaceutical compositions useful for treating a subject infected with Gram-
negative
bacteria. The method involves placing an effective amount of at least one
isolated
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polypeptide that binds selectively to at least a portion of an outer membrane
protein selected
from OmpA, PAL, MLP, in a pharmaceutically suitable carrier.
As used herein, the term "subject" refers to a vertebrate. In certain
embodiments the
subject is a human.
A "subject infected with Gram-negative bacteria" refers to a subject in which
living
Gram-negative bacteria have breached normal anatomic and functional protective
barriers
(e.g., skin, mucosa, etc.) and survived to multiply in a tissue, fluid, or
space within the subject
that is normally sterile. Typically, but not necessarily, Gram-negative
bacteria can be
cultured from infected tissue or body fluid obtained from a subject infected
with Gram-
negative bacteria. A "subject infected with Gram-negative bacteria" may have,
but need not
have, Gram-negative sepis.
As used herein, the term "Gram-negative bacteria" refers to bacteria that are
known in
the art as members of the Enterobacteriaceae, non-enteric Gram-negative
bacteria, and
anaerobic Gram-negative bacteria. These include but are not limited to the
following:
Enterobacteriaceae - Buttiauxella spp., Cedeca spp., Cedecea spp., Citrobacter
spp.,
Edwardsiella spp., Enterobacter spp., Escherichia spp., Ewingella spp., Hafnia
spp.,
Klebsiella spp., Kluyvera spp., Leclercia spp., Leminorell spp., Moellerella
spp., Morganella
spp., Obesumbacterium spp., Proteus spp., Providencia spp., Rhanella spp.,
Salmonella spp.,
Serratia spp., Shigella spp., Trabulsiella spp., Tutamella spp., Xenorhabdus
spp., Yersinia
spp., Yokenella spp., (and various "enteric groups" that are not as yet
assigned).
Non-enteric Gram-negative bacteria - Acinetobacter spp., Achromobacter spp.,
Actinobacillus spp., Aeromonas spp., Alcaligenes spp., Arcobacter spp.,
Bordetella spp
Borrelia spp., Branhamella spp., Brucella spp., Campylobacter spp.,
Capnocytophaga spp.,
Cardiobacterium spp., Chromobacterium spp., Commamonas spp., Eikenella spp.,
Flavimonas spp., Francisella spp., Haemophilus spp., Helicobacter spp.,
Kingella spp.,
Legionella spp., Moraxella spp., Neisseria spp., Ochrobactrum spp., Oligella
spp.,
Pasteruella spp., Plesiomonas spp., Protomonas spp., Pseudomonas spp.,
Sphingobacterium
spp., Streptobacillus spp., Vibrio spp., Weeksell spp., Xanthomonas spp.,
Yersinia spp.
Anaerobic Gram-negative bacteria - Bacteroides spp., Fusobacterium spp.
The invention also embraces isolated polypeptides capable of binding
selectively to at
least a portion of an OMP selected from OmpA, PAL, or MLP. Such polypeptides
can
include, for example, antibodies or fragments of antibodies ("binding
polypeptides").
Antibodies include monoclonal and polyclonal antibodies, prepared according to
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conventional methodology. See, e.g., Harlow & Lane, "Antibodies: A Laboratory
Manual,"
Cold Spring Harbor Laboratory, 1988.
The term "antibody" as used herein means at least a portion of an
immunoglobulin
molecule (see W. E. Paul, ed., "Fundamental Immunology," Lippincott-Raven,
Philadelphia,
1999, pp. 37-74) capable of binding to an antigen. Preferably the antibody
belongs to the
immunoglobulin G (IgG) class of antibodies. According to this definition, the
term
"antibody" includes not only intact antibodies but also various forms of
modified or altered
antibodies, such as an Fv fragment containing only the light and heavy chain
variable regions,
an Fab or (Fab)'2 fragment containing the variable regions and parts of the
constant regions, a
single-chain antibody, and the like. Bird et al., Science 242:424-426 (1988);
Huston et al.,
Proc Natl Acad Sci USA 85:5879-5883 (1988). The antibody may be of animal
(especially
mouse or rat) or human origin or may be chimeric (Morrison S et al., Proc Natl
Acad Sci USA
81:6851-6855 (1984)) or humanized (Jones et al., Nature 321:522-525 (1986),
and published
UK patent application 8707252). Methods of producing antibodies suitable for
use in the
present invention are well known to those skilled in the art and can be found
described in
such publications as Harlow & Lane, "Antibodies: A Laboratory Manual," Cold
Spring
Harbor Laboratory, 1988. The genes encoding the antibody chains may be cloned
in cDNA
genomic form by any cloning procedure known to those skilled in the art. See
for example
Maniatis et al., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor
Laboratory,
1982.
In another embodiment a pharmaceutical composition for use in treating a
subject
infected with Gram negative bacteria can include an isolated polyclonal
antibody that binds
specifically to at least a portion of OmpA, PAL, or MLP, prepared in a
pharmaceutically
suitable carrier. The binding interaction and the effects of such binding
between the
pharmaceutical composition and the outer membrane protein will be as just
described above
in reference to an isolated polypeptide that binds specifically to at least a
portion of OmpA,
PAL, or MLP.
According to this aspect of the invention, the polyclonal antibody binds to
OmpA,
PAL, MLP, or any combination of these OMPs, but not to at least one other
component
bound by JS antiserum. For example, the polyclonal antibody of the invention
can in some
embodiments bind to OmpA, PAL, or MLP, but not to other LPS-associated
lipoproteins.
In this aspect of the invention, the polyclonal antibody is raised by
immunizing an
animal, preferably a mammal, with an effective amount of isolated OmpA, PAL,
MLP, or
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any combination of these OMPs. The polyclonal antibody so prepared differs
from JS
antiserum insofar as the latter is raised against heat-killed whole bacteria
and thus binds to
antigens in addition to those related only to OmpA, PAL, and MLP.
In a particular embodiment of this aspect of the invention, the polyclonal
antibody can
be raised by immunizing a human with an effective amount of isolated OmpA,
PAL, MLP, or
any combination of these OMPs. The resulting human antiserum can be used
effectively in
human subjects.
Binding polypeptides that bind selectively to certain OMPs also may be derived
from
sources other than antibody technology. For example, such polypeptide binding
agents can
be provided by degenerate peptide libraries which can be readily prepared in
solution, in
immobilized form, as bacterial flagella peptide display libraries or as phage
display libraries.
Combinatorial libraries also can be synthesized of peptides containing one or
more amino
acids. Libraries further can be synthesized of peptides and non-peptide
synthetic moieties.
Phage display can be particularly effective in identifying binding peptides
useful
according to the invention. Briefly, one prepares a phage library (using,
e.g., m13, fd, or
lambda phage), displaying inserts from 4 to about 80 amino acid residues using
conventional
procedures. The inserts may represent, for example, a completely degenerate or
biased array.
One then can select phage-bearing inserts which bind to the OMP or a complex
containing an
OMP. This process can be repeated through several cycles of reselection of
phage that bind
to the OMP or complex. Repeated rounds lead to enrichment of phage bearing
particular
sequences. DNA sequence analysis can be conducted to identify the sequences of
the
expressed polypeptides. The minimal linear portion of the sequence that binds
to the OMP or
complex can be determined. One can repeat the procedure using a biased library
containing
inserts containing part or all of the minimal linear portion plus one or more
additional
degenerate residues upstream or downstream thereof. Yeast two-hybrid screening
methods
also may be used to identify polypeptides that bind to the OMPs. Thus, the
OMPs of the
invention, or a fragment thereof, or complexes of OMP can be used to screen
peptide
libraries, including phage display libraries, to identify and select peptide
binding polypeptides
that selectively bind to the OMPs of the invention. Such molecules can be
used, as described,
for screening assays, for purification protocols, for interfering directly
with the functioning of
OMPs and for other purposes that will be apparent to those of ordinary skill
in the art.
OmpA, PAL, MLP, or a fragment thereof, also can be used to isolate naturally
occurring polypeptide binding partners which may associate with the OMPs in
vitro or in
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vivo. Recently it has come to be appreciated that certain Toll-like receptors
(TLRs) are
responsible for cellular response to microbial products, including LPS and
lipoproteins.
Hirschfeld M et al., Jlmmunol 165:618-622 (2000). TLR4 and TLR2 have been
associated
with LPS signaling, and a point mutation in the tlr4 gene in C3H/HeJ mice has
been reported
to account for the observed hyporesponsiveness of that strain to LPS. Thus
OmpA, PAL, and
MLP may be useful, for example, in further elucidating the details of TLR-
mediated
signaling as well as other receptors and pathways involved in LPS signaling.
Isolation of
binding partners may be performed according to well-known methods. For
example, isolated
OmpA, PAL, or MLP can be attached to a substrate, and then a solution
suspected of
containing an OMP-binding partner may be applied to the substrate. If the
binding partner
for OmpA, PAL, or MLP is present in the solution, then it will bind to the
substrate-bound
OMP. The binding partner then may be isolated for identification and further
study. Other
proteins which are binding partners for OmpA, PAL, or MLP, may be isolated by
similar
methods without undue experimentation.
The invention in another aspect provides an immortal cell line which secretes
a
polypeptide that binds specifically to OmpA, PAL, MLP, or immunogenic portions
thereof.
Preferably, the secreted polypeptide is a monoclonal antibody directed against
OmpA, PAL,
or MLP. In alternative embodiments the secreted polypeptide can also be a
fragment of a
monoclonal antibody directed against OmpA, PAL, or MLP, or it can be a fusion
protein
incorporating an antigen-binding portion of such an antibody.
As used herein, "immortal cell line" refers to a hybridoma, myeloma, or a
transfected
cell line that, under proper conditions, can be propagated indefinitely. In a
preferred
embodiment the immortal cell line is a hybridoma prepared by cell fusion
between
splenocytes from an immunized animal and a myeloma according to standard
techniques.
Kohler G et al., Eur J Immunol 6:292-295 ( 1976). In another embodiment the
immortal cell
line can be a myeloma or non-immune cell that is transfected with a nucleic
acid that
operably encodes an antibody, antibody fragment, fusion protein, or the like.
In yet another
embodiment the immortal cell line can be a myeloma or hybridoma that is
directed to express
a desired polypeptide through homologous recombination.
The term "secretes" as used herein refers to expression of polypeptide in a
form that
can be isolated for the purposes of the invention. In the instance of a
hybridoma, the
polypeptide typically is expressed and released into the medium in which the
hybridoma is
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grown. Forms of expression that result in polypeptides that remain associated
with the cell
membrane or that remain in an intracellular compartment are also encompassed
by the use of
this term.
In yet another aspect he invention further provides a method of actively
immunizing a
subj ect against infection due to Gram-negative bacteria. The method involves
administering
to a subject an isolated OMP antigen selected from OmpA, PAL, MLP, or an
immunogenic
portion thereof, prepared in a pharmaceutically suitable carrier, in an amount
effective for
inducing protection of the subject against infection due to Gram-negative
bacteria.. The
method can entail immunization against any one or any combination of the three
OMP
antigens, and it can further entail administration of the OMP antigen with an
adjuvant that is
distinct from OmpA, PAL, or MLP. Examples of such adjuvants are listed above.
In this
context, an effective amount is that amount sufficient to induce a protective
immune response
to the antigen. This can be manifest as a titer of circulating IgG antibody
specific for the
antigen which is at least about 1:16 or at least twice that of a control titer
as measured in an
unexposed nonimmune subject. Alternatively, it can be manifest as a prompt
anamnestic
response (with increase in antigen-specific IgG titer) upon reexposure to the
antigen.
The term "antigen" broadly includes any type of molecule, typically a
polypeptide or
polysaccharide, which is recognized by a host immune system as being foreign.
An "OMP
antigen" as used herein refers to any intact form or immunogenic fragment of
OmpA, P.AL,,
or MLP that can induce a immune response specific to that OMP. A specific
immune
response typically involves the generation of antibodies that bind
specifically to at least one
epitope of the antigen. A specific immune response can also involve the
response by T cells
bearing antigen receptors that specifically recognize peptide fragments of an
antigen in
association with major histocompatibility complex (MHC). Thus a specific
immune response
to an OMP antigen can include the generation of antibodies that bind
specifically to at least
one epitope of the OMP antigen and the response by T cells bearing antigen
receptors that
specifically recognize peptide fragments of an OMP antigen in association with
MHC.
The invention in another aspect provides a method of treating a subject who
has an
infection with Gram-negative bacteria. The method involves administering to a
subject in
need of such treatment an isolated polypeptide that binds specifically to
OmpA, PAL, or
MLP in an amount effective to treat the infection with the Gram-negative
bacteria.
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Preferably the isolated polypeptide is administered in an amount effective to
inhibit growth of
the Gram-negative bacteria in vivo. This inhibitory effect on growth can be
determined by
methods well known in the art, including , e.g., comparing the number of
colony-forming
units in a standard culture taken from an infected body fluid in the presence
of and in the
absence of the polypeptide. An inhibitory effect due to the presence of the
polypeptide would
be associated with a diminished or declining number of colonies in comparison
to the
corresponding number of colonies in the absence of the polypeptide. More
preferably the
isolated polypeptide is administered in an amount effective to inhibit Gram-
negative sepsis.
The isolated polypeptide can be an antibody or another polypeptide (as
described above), so
long as it binds specifically to OmpA, PAL, or MLP in vivo. The isolated
polypeptide is
administered in a pharmaceutically suitable carrier.
The term "treating" is defined as administering, to a subject, a
therapeutically
effective amount of a compound that is sufficient to prevent the onset of,
alleviate the
symptoms of, or stop the progression of a disorder or disease being treated.
The phrase
"therapeutically effective amount" means that amount of a compound which
prevents the
onset of, alleviates the symptoms of, or stops the progression of a disorder
or disease being
treated. Thus, as used herein, an amount effective to treat an infection
caused by Gram-
negative bacteria is an amount effective to prevent the onset of, alleviate
the symptoms of, or
stop the progression of an infection caused by Gram-negative bacteria.
As used herein, the term "inhibit Gram-negative sepsis" refers to inhibition
of any
aspect of the multitude of inducing and responding signals and events which
are associated
with the systemic inflammatory response to infection with Gram-negative
bacteria. This is
meant to encompass both early and late sepsis, i.e., both before and during
the stage with
cardiovascular decompensation and end organ dysfunction and injury. Early
events and
signals in the development of sepsis can include induction of proinflammatory
cytokines,
e.g., IL-1 [3, IL-6, and TNF-a,, as well as elaboration and release of other
cytokines and
mediators, including IL-8, gamma interferon (IFN-y), chemokines, migration
inhibitory factor
(MIF), nitric oxide, kinins, complement, platelet activating factor (PAF),
etc. Organs
particularly susceptible to sepsis-related dysfunction and injury in late
sepsis include lung,
liver, and kidneys. Other problems frequently encountered in late sepsis
include dysfunction
of the skin, gastrointestinal tract, central nervous system, bone marrow, and
cardiovascular
system.
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Without meaning to be bound by any particular theory, the mechanisms by which
the
inhibition of Gram-negative sepsis is believed to be achieved include
clearance,
neutralization, and opsonization. These various mechanisms can be applied to
whole
bacteria, insoluble fragments of bacteria, and soluble factors released from
bacteria. Soluble
factors released from bacteria include the OMPs themselves, either free or in
complexes with
LPS.
The term "clearance" as used herein refers to removal from the circulation.
This can
include clearance by excretion, sequestration, degradation, and the like.
"Insoluble fragments of Gram-negative bacteria" as used herein refers to any
particulate component or aggregate of components originating from Gram-
negative bacteria
which can be precipitated out of serum or out of solution by centrifugation.
Examples of
such fragments include cell wall fragments, membrane blebs, etc.
The term "neutralize" as used herein refers to the abrogation of biological
activity of a
molecule by steric interference of the interaction between the biologically
active molecule
and its cellular receptor. As applied to whole bacteria, the term "neutralize"
refers to
abrogation of biological activity of whole bacteria by steric interference of
the interaction
between the biologically active molecules on the bacteria and their receptors
on cells of an
infected host. Similarly, as applied to insoluble fragments of bacteria, the
term "neutralize"
refers to abrogation of biological activity of the fragments by steric
interference of the
interaction between the biologically active molecules on the fragments and
their receptors on
cells of an infected host.
The term "opsonize" as used herein refers to the formation of immune complexes
between antibodies and their cognate antigens. Opsonization can result in
phagocytosis of
the bound target, elimination of the bound target from the circulation, and
neutralization. In
relation to whole bacteria, opsonization also can lead to cell lysis through
complement
activation.
According to this aspect of the invention, the method of treating a subject
who has
Gram-negative sepsis may further include administering to the subject an
effective amount of
an immune stimulant. An immune stimulant can include an adjuvant (described
above), a
cytokine, or a substance that induces a cytokine or costimulatory molecule.
Cytokines include interleukins, interferons, certain growth factors, and
colony
stimulating factors. Included among these are, e.g., interleukin (IL)-2, IL-4,
IL-6, IL-10, IL
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12, interferon (IFN)-y, tumor necrosis factor (TNF)-a,, transforming growth
factor (TGF)-(3,
and granulocyte colony stimulating factor (G-CSF).
Costimulatory molecules include, for example, CD2, CD28, CD40, CD48, CD80 (B7-
1 ), CD86 (B7-2), CD 152 (CTLA-4).
Chemokines include compounds in four subfamilies based on their structure:
CXC,
CC, C, and CX3C. Examples of chemokines include MIP-la, MIP-1 (3, RANTES, MCP-
1,
MCP-2, IL-8, and GROoc, among others.
Assays for immunoglobulins, cytokines, costimulatory molecules, and chemokines
are well known to those skilled in the art. See, e.g., Current Protocols in
Molecular Biology,
John Wiley & Sons, New York, 1999. A number of commercial kits, particularly
ELISAs,
are available for most of these secreted products.
In yet another aspect the invention provides a method of treating a subject
who has
Gram-negative sepsis. The method involves administering an isolated
polypeptide that binds
specifically to OmpA, PAL, or MLP in an amount effective to inhibit sepsis-
related release of
at least one soluble factor into blood or tissue of the subject. The isolated
polypeptide that
binds specifically to at least a portion of OmpA, PAL, or MLP can include an
antibody, a
fragment of an antibody, or another polypeptide as described above.
An amount effective to inhibit sepsis-related release of at least one soluble
factor into
blood or tissue of the subject is an amount that, when given to a subject
under conditions
where the at least one soluble factor is normally released into blood or
tissue in the absence
of the inhibitor, is sufficient to prevent release or decrease the amount
released of the at least
one soluble factor in the blood or tissue in the presence of the polypeptide.
Soluble factors
released in relation to sepsis can include factors originating from the
infective bacteria or
from the host. Examples of soluble factors released from Gram-negative
bacteria include
OMPs, LPS, and free lipids. Examples of soluble factors of host origin include
cytokines
(e.g., IL-l, IL-6, TNF-oc), HMG-1 (Wang H et al., Science 285:248-251 (1999)),
chemokines,
MIF, and nitric oxide.
The invention further provides a method of treating a subject who has Gram-
negative
sepsis. The method involves administering to a subject with Gram-negative
sepsis an isolated
polypeptide that binds specifically to at least a portion of OmpA, PAL, or MLP
in an amount
effective to enhance clearance of at least one sepsis-related soluble factor
released by Gram-
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negative bacteria into blood of the subject. The isolated polypeptide that
binds specifically to
at least a portion of OmpA, PAL, or MLP can include an antibody, a fragment of
an antibody,
or another polypeptide as described above. In a preferred embodiment, the
polypeptide is a
monoclonal antibody specific for OmpA, PAL, or MLP. A sepsis-related soluble
factor
released by Gram-negative bacteria into blood of the subject can include any
one or
combination of the following: LPS, OmpA, PAL, and MLP.
The present invention is further illustrated by the following Examples, which
in no
way should be construed as further limiting. The entire contents of all of the
references
(including literature references, issued patents, and published patent
applications) cited
throughout this application are hereby expressly incorporated by reference.
Examples
Bacterial strains, media, and growth conditions. E. coli JS was the kind gift
of J.C.
Sadoff (Walter Reed Army Institute of Research, Washington, DC). E coli
018:K1:H7
strain Bort (designated E coli 018K+), E. coli 018:K1-:G2A (a nonencapsulated
derivative
of 018:K1:H7, designated E. coli 018K-), E. coli 08:K45:H1, E. coli 016:K1:H6,
and E.
coli 025:KS:H1 were kind gifts of A. Cross (University of Maryland Cancer
Center,
Baltimore). OMP-deficient E. coli K12 and E. coli 018 mutants and closely
related OMP-
containing bacteria were used for immunoblotting studies. E. coli 018 E91
(OmpA-deficient
derivative of E coli Ol 8:K1:H7) and E69 (OmpA-restored derivative of E. coli
018:K1:H7)
were kind gifts of K.S. Kim (Los Angeles Children's Hospital). Prasadarao NV
et al., Infect
Immun 64:146-153 (1996). E. coli K12 1292 (Lazzaroni J-C and Portalier R, Mol
Microbiol
6:735-742 (1992)), JC7752 (PAL-deficient derivative of 1292), and 7752p417
(PAL-restored
mutant of JC7752) were kindly provided by J.-C. Lazzaroni (Universite Claude
Bernard,
Lyon 1, France). E. coli K12 p400, CH202 (PAL-deficient mutant of p400), and
CH202(pRC2) (PAL-restored derivative of CH202) were kindly provided by U.
Henning
(Max-Planck-Institut fiir Biologie, Tiibingen, Germany). Chen R and Henning U,
Eur J
Biochem 163:73-77 (1987).
E. coli K12 AT1360 (Lpp+; mutations: DE [gpt-proA] 62, lacyl, tsx-29, g1nV44
[AS],
galK2 [0c], LAM-, aroD6, hisG4 [0c], xylAS, mtl-1, argE3 [0c], thi-1) and E
coli K12
JE5505 (L pp ; mutations: DE [gpt-proA] 62, lacyl, tsx-29, g1nV44 [AS], galK2
[0c], LAM-,
lpp-254 [del], pps-6, hisG4 [0c], xylAS, mtl-1, argE3 [0c], thi-1) were
obtained from the E.
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coli Genetic Stock Center (New Haven, CT). Pittard J and Wallace BJ,
JBacteriol 91:1494-
1500 (1966); Hirota Y et al., Proc Natl Acad Sci USA 74:1417-1420 (1977).
Although not
isogenic, these two mutants have nearly identical mutation profiles, and
differ only in: lpp
(the gene encoding murein lipoprotein is deleted in E. coli K12 JE5505), aroD6
(the gene
encoding 3-dehydroquinase, a 26 kDa protein, is mutated in the Lpp+ strain
(Duncan K et al.,
Biochem J238:475-483 (1986)), and pps-6 (the gene encoding phosphenolpyruvate
synthase,
a roughly 84 kDa protein is mutated in the Lpp strain (Geerse RH et al., Mol
Gen Genet
218:348-352 (1989)).
Bacteria were cultured in trypticase soy broth (TSB, Difco, Detroit) from
colonies
stored on trypticase soy agar (TSA, Difco). Media was supplemented with
kanamycin (50
mg/ml) for E. coli K12 CH202pRC2 and ampicillin (100 mg/ml) for E. coli K12
JC7752p417
to maintain the plasmids. Bacteria were cultured at 37 °C with vigorous
agitation to the
desired growth phase, harvested, and washed by low speed centrifugation in
sterile normal
saline (5000-8000 x g, 8-10 minutes, 4 °C).
Example 1
Monoclonal antibodies
Methods. Prior studies indicated that anti-JS IgG binds three OMPs of MWs 35
kDa,
18 kDa (previously estimated as 37 kDa and 24 kDa respectively: Hellman J et
al., Jlnfect
Dis 176:1260-1268 (1997)) and 5-9 kDa, that are present on the bacterial
surface and are
released into human serum as OMP/LPS complexes. Monoclonal antibodies were
prepared
against each of the three OMPs bound by IgG in JS antiserum, and against the O-
polysaccharide of E. coli 018 LPS. For production of anti-OMP monoclonal
antibodies,
BALB/c mice (Charles River Laboratories, Wilmington, MA) were immunized with
heat-
killed, lyophilized E. coli JS vaccine prepared as described. Siber GR et al.,
Jlnfect Dis
152:954-964 (1985). Vaccine was resuspended in sterile normal saline (1
mg/ml).
Increasing doses were injected intraperitoneally 3 times per week for three
weeks (0.1 mg,
0.2 mg, and 0.3 mg). Booster injections were given monthly for 1-3 months,
with the final
booster three days prior to harvesting the spleen. Splenocytes were harvested
and fused with
myeloma cells by standard laboratory protocol. Kohler G et al., Eur Jlmmunol
6:292-295
(1976); Cold Spring Harbor Laboratory (1988) Antibodies: A Laboratory Manual,
Cold
Spring Harbor Press, Cold Spring Harbor. Fused cells were cultured in
Dulbecco's
Modification of Eagle's Medium (DMEM, Mediatech Cellgro) supplemented with
glucose
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(4.5 gm/L), L-glutamine, 20% heat-inactivated fetal calf serum (Mediatech),
penicillin ( 100
units/ml), and streptomycin (100 mg/ml).
The three OMPs are exposed on the surface of bacteria after incubation in
human
serum. Hellman J et al., Jlnfect Dis 176:1260-1268 (1997). Accordingly,
monoclonal
antibodies were initially screened by bacterial ELISA, using heterologous
serum-exposed
smooth E. coli isolates (E. coli 08:K45:H1, 016:K1:H6, and 025:K5:H1) as the
coating
antigen, and hybridoma culture supernatants as primary antibody. Hellman J et
al., J Inf Dis
176:1260-68 (1997). Bacteria were grown to the desired phase as determined by
optical
density at 550 nm (Asso), washed in sterile saline, suspended in serum or
saline to an ASSO of
1.0, and incubated at 37 °C for the specified time (10 minutes -1
hour). The bacteria were
washed by centrifugation (5000-8000 x g, 8-10 minutes, 4 °C) three
times in sterile normal
saline and resuspended in an equal volume of carbonate buffer, pH 9.6 (50 mM
sodium
carbonate; EM Science, Cherry Hill, NJ). Polyvinyl microtiter plates (Dynatech
Laboratories, Chantilly, VA) were coated with bacteria (10g bacteria/ml) and
incubated
overnight at 4 °C. The microtiter plates were then washed three times
(PBS, 1 mg/ml Tween
20, 1 mg/ml bovine serum albumin [BSA], 2 mg/ml MgCl2), blocked overnight at 4
°C with
PBS containing BSA (1 mg/ml), and washed again. Dilutions of either normal
rabbit serum
(NRS) or rabbit antiserum to E coli J5 were added and plates were incubated (2
hours, 37
°C). After three additional washings, horseradish peroxidase-conjugated
anti-rabbit IgG
(Cappel, Durham, NC) was added, and the plates were incubated (2 hours, 37
°C) and
washed. Peroxidase substrate (1 mg/ml H202 in ABTS, citric acid, Na2HP04) was
added,
plates were incubated at room temperature for 30 minutes, and the A4o5 was
read (ELISA
reader EAR400; SLT Lab Instruments, Hillsborough, NC). Titers were determined
using a
standard curve as previously described. Zollinger WD and Boslego JW, Jlmmunol
Methods
46:129-140 (1981). Standard curves were generated using known concentrations
of rabbit
IgG (Cappel). All assays were performed in duplicate and mean values
determined.
Antibodies that bound to serum-exposed bacteria were then analyzed for binding
to
the three OMPs by immunoblotting using E. coli 025:K5:H1 bacterial lysates as
antigen and
supernatants from fusions as primary antibody. Immunoblotting was used to
detect binding
of antisera and monoclonal antibodies to washed bacteria (106/well) and
bacterial antigens
that were affinity purified from filtrates of serum exposed bacteria. All
samples were
prepared in sample buffer (2.5% SDS, 22% glycerol, 0.5% (3-mercaptoethanol,
and trace
bromophenol blue in Tris base). Samples were electrophoresed on 16% SDS-
polyacrylamide
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gels and transferred to nitrocellulose (Bio-Rad Laboratories, Hercules, CA) by
applying 200
mA of constant current at 4 °C for 1 hour (Hoefer Scientific
Instruments, San Francisco). For
most experiments, the nitrocellulose was blocked (1 hour at room temperature,
or overnight
at 4 °C) with 1% powdered skim milk in TTBS (150 mM NaCI, 50 mM Tris,
0.1% Tween-
20, pH 7.5), washed for 10-15 minutes with TTBS, incubated with primary
antibodies, and
washed 3 times. Primary antibodies included IgG in rabbit antisera to heat-
killed E. coli JS
and E. coli 018 O-polysaccharide (both diluted 1:500 in TTBS), IgG in mouse
antiserum to
heat-killed E coli J5, and marine monoclonal antibodies directed to each of
the three OMPs
(at a concentration of 1 ~g/ml). Blots were then incubated for 30 minutes with
biotin-
conjugated anti-rabbit or anti-mouse IgG antibody (Vectastain, Vector
Laboratories,
Burlingame, CA) diluted 1:240 in TTBS, washed, and then incubated for 30
minutes in a
mixture of avidin and biotinylated horseradish peroxidase complex, as
described in the
manufacturer's instructions (Vectastain). After a final wash with PBS,
peroxidase substrate
was added (2 ml of 3 mg/ml 4-chloro-1-naphthol, 8 ml of PBS, 10 microliters of
30% H2O2).
The reaction was stopped after 30 minutes by repeated rinsing with distilled
water.
Following initial screening, hybridomas of interest were subcloned by limiting
dilution to one cell in every fourth well to derive subclones with strong
growth characteristics
and high production of the antibodies with the binding characteristics
described below.
Polyclonal mouse anti-JS IgG was used as a positive control, and pre-immune
serum served
as the negative control.
Two methods were used to prepare large amounts of the monoclonal IgGs from the
hybridoma cell lines isolated as described above. Monoclonal antibodies
directed to each of
the three OMPs and to the O-polysaccharide of E. coli 018 LPS (Mab anti-018
IgG) were
produced in ascites of BALB/c mice by mouse hybridoma cell lines. The
hybridoma cell line
producing Mab anti-018 IgG was the kind gift of A. Cross. Kim KS et al.,
Jlnfect Dis
157:47-53 (1988). Ten days after intraperitoneal instillation of 0.5 ml of
Pristane (Sigma, St.
Louis, MO), 5-10 x 106 hybridoma cells were collected, washed twice in Hanks'
Balanced
Salt Solution (Cellgro, Mediatech Inc., Herndon, VA), and injected
intraperitoneally. Ascites
was collected by aspiration every 2-3 days three times. Cold Spring Harbor
Laboratory
(1988) Antibodies: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring
Harbor.
Monoclonal antibody against the 18 kDa OMP was also produced in an artificial
capillary
cell culture system (Cellmax, Cellco, Laguna Hills, CA). The cartridge
(Cellmax 011
module) was inoculated with 2.Sx10'viable cells. Culture medium was Dulbecco's
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Modification of Eagle's Medium (DMEM, Mediatech Cellgro) supplemented with
glucose
(4.5 gm/L), L-glutamine, 2.5-10% heat-inactivated fetal calf serum
(Mediatech), penicillin
(100 units/ml), and streptomycin (100 mg/ml). The concentration of IgG
produced in the
artificial capillary cell culture was 0.3-1.0 mg/ml as determined by ELISA.
Anti-OMP
antibodies showed no cross-reactivity with LPS or with proteins in human serum
by
immunoblotting. The Mab anti-018 IgG does not cross-react with LPS from other
organisms, with the OMPs, or with proteins in human serum by immunoblotting.
IgG was purified from ascites following ammonium sulfate precipitation and
from
hyperimmune serum. Cold Spring Harbor Laboratory (1988) Antibodies: A
Laboratory
Manual, Cold Spring Harbor Press, Cold Spring Harbor; Warren HS et al.,
Jlnfect Dis
163:1256-1266 (1991); Ge Y et al., Jlnfect Dis 169:95-104 (1994). Briefly,
affinity
chromatography was performed by passage over a protein G-Sepharose 4 fast-flow
column
(Pharmacia, Piscataway, NJ). Bound IgG was eluted from the column with 0.1 M
glycine
(pH 2.7) and was immediately neutralized using 1 M Tris buffer (pH 9.0).
Purified IgG was
dialyzed against PBS (pH 7.2) and stored at -80 °C. Protein
concentration was determined by
ELISA and by absorption at 280 nm. Zollinger WD and Boslego JW, Jlmmunol
Methods
46:129-140 (1981).
Results. Of 10 splenic fusions, 9 antibodies were identified that bound to the
surface
of heterologous serum-exposed bacteria by ELISA. Immunoblotting analysis
revealed that 7
of the 9 IgGs bound to one of the three OMPs. Three of these anti-OMP
monoclonal IgGs
(2D3, 6D7, and 1 C7) were selected for increased production, each with
specificity for one of
the three OMPs. A representative immunoblot of lysates of E. coli 06 bacteria
stained with
these three monoclonal IgGs and polyclonal anti-JS IgG is shown in Figure 1.
Antigen was electrophoresed on a 16% SDS-polyacrylamide gel and transferred to
nitrocellulose. Primary antibodies for the immunoblots include polyclonal
mouse anti-JS IgG
(lane 1), and three separate monoclonal antibodies, 2D3 (lane 2), 6D7 (lane
3), and 1C7 (lane
4) derived from mice immunized with E. coli JS vaccine. Estimated molecular
weights of the
bands (kDa) are indicated at the left of the figure.
Example 2
Identification of OmpA
We hypothesized that the 35 kDa protein was OmpA based upon the apparent
molecular weight and the fact that the electrophoretic mobility of the band
was altered by
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boiling. Hindennach I and Henning U, Eur JBiochem 59:207-213 (1975).
Immunoblotting
studies were performed to identify this protein.
Recombinant outer membrane protein A (OmpA). The coding region of the 325
amino acid mature OmpA protein, excluding the 21 amino acid signal sequence
(GenBank
accession #V00307), was generated by PCR amplification of DNA from an extract
of E. coli
018:K1:H7. OmpA-specific PCR primers OmpABacl and OmpABac2 contained 5'
extensions for cloning into the transfer plasmid pBACgus-2cp (Novagen,
Madison, WI).
OmpABacl: 5'-GACGACGACAAGGCTCCGAAAGATAACACCTG-3' (SEQ ID NO:1)
OmpABac2: 5'-GAGGAGAAGCCCGGTTAAGCCTGCGGCTGAGTTAC-3' (SEQ ID N0:2)
The transfer plasmid containing the OmpA coding sequence (OmpA/pBACgus-2cp)
was then
transfected into the BacVector-2000 Triple Cut Baculovirus DNA in Sf~ cells,
according to
the manufacturer's instructions (Novagen, Madison, WI). Positive recombinants
were
expanded, and high titer virus was produced, to give multiplicity of infection
in the range of
10 to 20 for maximal protein expression in Sf9 cells. The final Baculovirus
construct
contained the OmpA coding sequence, with an in-frame amino terminal extension
(fusion
sequences were encoded by the pBACgus-2cp transfer plasmid) containing an
enterokinase
recognition sequence, an S-protein binding site and a polyhistidine tail. The
36.5 kDa OmpA
fusion protein (calculated molecular weight) was purified from Baculovirus-
infected Sf~3 cell
lysates by polyhistidine affinity chromatography over a Talon cobalt metal
affinity resin
according to the manufacturer's instructions (Clontech, Palo Alto, CA).
The 35 kDa OMP is OmpA. Isolates of E. coli Ol 8 bacteria in which the OmpA
gene
was deleted and then replaced back into the strain (Prasadarao NV et al.,
Infect Immun
64:146-153 (1996)) and recombinant OmpA were electrophoresed on 16% SDS-
polyacrylamide gels, transferred to nitrocellulose, and used as antigen in
immunoblotting
assay performed as described above. Primary staining antibodies included anti-
JS IgG and
monoclonal IgG that is directed against the 35 kDa OMP (2D3). Anti-JS IgG and
2D3 did
not react with the 35 kDa band in lysates of bacteria in which the OmpA gene
was deleted,
but did react with a 35 kDa band in the wild-type strain and the strain in
which the gene was
reinserted (Figure 2). Bacterial strains are: wild type OmpA+E coli 018:K1:H7
(lane 1);
E91, an OmpA-deleted mutant of E. coli 018:K1:H7 (lane 2); E69, and an OmpA-
restored
mutant of E coli 018:K1:H7 (lane 3). Molecular weight maxkers (kDa) are as at
the left.
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Recombinant OmpA was stained by anti-JS IgG and 2D3 (Figure 3). Recombinant
OmpA (lane 1 of each panel) and lysates of E. coli 018:K1:H7 bacteria (lane 2
of each panel)
were electrophoresed on a 16% SDS-polyacrylamide gel and transferred to
nitrocellulose.
Primary antibodies included polyclonal mouse anti-JS IgG (left panel) and the
monoclonal
antibody 2D3 (right panel). Recombinant OmpA ran at a slightly higher
molecular weight,
presumably because of the polyhistidine tag that is present on the recombinant
protein. These
results indicate that the 35 kDa OMP is OmpA and that 2D3 is a monoclonal anti-
OmpA IgG.
Example 3
Identification of PAL
Methods. The final purification procedure for the 18 kDa OMP consisted of: 1 )
preparation of total bacterial membranes, 2) Triton X-100 extraction of
bacterial membranes,
3) affinity chromatography using sepharose beads conjugated with 6D7 (the anti-
18 kDa
OMP monoclonal antibody), and 4) reverse-phase HPLC separation. The
purification steps
are described below.
Total bacterial membranes were prepared from mid-late log-phase cultures of E.
coli
018K- bacteria essentially as described. Hellman J et al., Jlnfect Dis
176:1260-1268 (1997);
Munford RS et al., JBacteriol 144:630-640 (1980). Unless otherwise indicated,
all steps
were performed at 4-6 °C. 2 L cultures of bacteria were harvested by
centrifugation and the
resultant pellets were resuspended in a total of 60 ml pre-chilled 10 mM HEPES
buffer (pH
7.4) with 25% sucrose (w/v) and 0.2 mM dithiothreitol (DTT, Fisher
Biochemicals, Fair
Lawn, NJ). RNase and DNase (Sigma, St. Louis) were each added to a final
concentration of
4 ~g/ml. Cells were disrupted by sonicating the suspension on ice (microtip,
30-60 second
bursts separated by 60-90 seconds, total sonication time 4 minutes). Unbroken
bacteria and
other debris were removed by centrifugation (10,000 x g, 40 minutes), and the
supernatant
was collected (volume 60 ml). 15 ml of HEPES buffer (pH 7.4) containing EDTA
(25 mM),
and DTT (0.2 mM) was added to the 60 ml to adjust the concentration of sucrose
to 20%
(w/v) and the concentration of EDTA to 5 mM. Samples were layered onto a 60%
(w/v)
sucrose cushion (7.5 ml sample per 4.5 ml cushion) and ultracentrifuged
(100,000 x g, 3
hours, 6 °C). Bacterial membranes present in the hazy white/yellow band
at the interface
were collected by puncturing the side of the tube with a 20 gauge needle and
aspirating gently
with a 1 ml syringe (approximately 0.5 ml/tube, final volume 5 ml). Total
membranes were
dialyzed against Tris-HCl (20 mM, pH 8) overnight (2 L) and then against fresh
buffer for 48
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hours (4 L). The final volume of dialyzed material was approximately 15 m1/2 L
of the
starting bacterial culture.
Sixty ml of dialyzed total membranes representing 8 liters of the starting
bacterial
culture were concentrated to 36 ml using a nitrogen pressurized system and a
Diaflo
ultrafiltration membrane, YM30 filter (Millipore Company, Danvers, MA)
according to the
manufacturer's instructions, and extracted with Triton X-100. Twelve ml of a
stock solution
of 10% Triton X-100 in Tris-HCl (20 mM, pH 8.4), containing the protease
inhibitor 4-(2-
aminoethyl)-benzenesulfonyl fluoride (Sigma) and EDTA were added to the
membranes
(final concentrations: 2.5% Triton X-100, 0.5 mM 4-(2-aminoethyl)-
benzenesulfonyl
fluoride, 5 mM EDTA). The sample was incubated at room temperature for 30
minutes, and
then ultracentrifuged (TH641 swinging bucket rotor, 100,000 x g, 2 hours, 6
°C). The
resultant supernatant (48 ml) was saved.
The detergent-extracted membrane supernatant was circulated overnight at 9-10
ml/hour through a 5.5 ml column of mouse monoclonal IgG (6D7) directed against
the 18
kDa OMP covalently conjugated to CNBr-activated Sepharose 4B beads (4
°C). Unbound
material was washed from the column with 36 ml of 2.5% Triton X-100 in 200 mM
NaPhos,
0.5 M NaCI, pH 6.8. Bound antigen was eluted with increasing concentrations of
SDS
(0.125, 0.25, 0.5, and 1 %, in 200 mM phos, 0.5 M NaCI, pH 6.8). Three
milliliters of each
concentration of SDS was applied to the column followed by 9 ml of wash
buffer. The
protein was detected in 0.5% and 1% SDS-eluted samples. Material eluted with
0.5% and
1% SDS were combined and concentrated to 4 ml by centrifugation in a Centricon
Plus-20
centrifugal filter device (10 kDa cutoff, Biomax-8 series, Millipore
Corporation).
Three milliliters of the concentrated affinity-purified sample was applied to
an
analytical C4 reverse-phase HPLC column (Vydac, Hesperia, CA) and eluted using
a linear
gradient of 5-95% acetonitrile/0.1% trifluoroacetic acid/HZO at a flow rate of
1 ml/min.
Fractions were collected at one minute intervals into 20 microliters of 2-fold
concentrated
SDS-PAGE sample buffer (5% SDS, 44% glycerol in Tris base) and lyophilized.
Lyophilized samples were resuspended in 40 microliters of water with (3-
mercaptoethanol
(0.5%) and trace bromophenol blue and heated (100 °C, 5-10 minutes).
Fractions were
electrophoresed and analyzed for the 18 kDa OMP by immunoblotting using anti-
JS IgG or
6D7 (the monoclonal anti-18 kDa OMP IgG) as the primary antibody.
The peak fraction from the C4 HPLC separation was electrophoresed on a 16% SDS-
polyacrylamide gel and stained with Coomassie brilliant blue. The faintly
staining 18 kDa
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band was then cut from the gel, washed twice (50% acetonitrile, 0.5 ml, 3
minutes) and
frozen. Sequence analysis of two peptides of a trypsin digestion of the
protein in the gel was
performed at the Harvard Microchemistry Facility by tandem mass spectrometry
(MS/MS) on
a Finnigan LCQ quadrupole ion trap mass spectrometer.
The 18 kDa OMP is PAL. Two peptide sequences (Sequence 1 and Sequence 2, 10
and 14 amino acids, respectively) were obtained that each mapped with 100%
homology to
PAL.
Sequence 1: VTVEGHADER (SEQ ID N0:3)
Sequence 2: [G][V]SADQ I VSYGK
* * * (SEQ ID N0:4)
Brackets ([ ]) indicate that the amino acid has been identified with
reasonable confidence.
Stars (*) indicate that the amino acid is isobaric and cannot by unambiguously
differentiated
by mass spectrometric sequencing. All other amino acids were assigned with the
highest
confidence.
The identity of PAL was confirmed by immunoblotting studies. Referring to
Figure
4, lysates of E. coli K12 bacteria in which the PAL gene (excC) was deleted,
or was deleted
and then replaced, were immunoblotted using with anti-JS IgG (left panel) or
monoclonal
anti-18 kDa OMP IgG 6D7 (right panel) as primary antibody. Bacterial strains
in both panels
of Figure 4 are: E. coli K12 p400 containing PAL (lane 1); CH202, a PAL-
deficient mutant
of E. coli K12 p400 (lane 2); CH202 prC2, a PAL-restored mutant of CH202 (lane
3); E. coli
K12 1292 containing PAL (lane 4); JC7752, a PAL-deficient mutant of 1292 (lane
5); and
JC7752 p417, a PAL-restored mutant of JC7752 (lane 6). Anti-JS IgG and 6D7 did
not react
with the 18 kDa band in lysates of PAL-deficient bacteria, but did react with
an 18 kDa band
in the wild-type strain and the strain with the gene reinserted (Figure 4).
These results
indicate that the 18 kDa OMP is PAL, and that 6D7 is a monoclonal anti-PAL
antibody.
Example 4
Identification of MLP
The 5-9 kDa OMP is murein lipoprotein (MLP). We hypothesized that the 5-9 kDa
OMP was MLP based on its low molecular weight and size heterogeneity. Hantke K
and
Braun V, Eur JBiochem 34:284-296 (1973). Accordingly, an isolate of E coli K12
in which
the murein lipoprotein (lpp) gene was deleted, a very closely related mutant
strain containing
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MLP, and the standard laboratory strain, E. coli 018 (also containing MLP),
were used as
antigens on identical immunoblots. Referring to Figure 5, the immunoblot in
the left panel
was developed with anti-JS IgG, and the immunoblot in the right panel was
developed with
the monoclonal IgG that binds to the 5-9 kDa OMP (1C7). Various lanes in the
two
immunoblots shown in Figure 5 correspond to: E. coli 018K+, containing MLP
(lane 1);
MLP-deficient E coli K12 JE5505 (lane 2); and closely related E coli
K12AT1360,
containing MLP (lane 3). Anti-JS IgG and 1C7 IgG did not react with the 5-9
kDa band in
bacterial lysates of the MLP-deficient strain (lane 2). These results
demonstrate that the
lower molecular weight cross-reactive OMP is MLP and that monoclonal antibody
1 C7 reacts
with MLP.
As mentioned above, the mutation profiles of the Lpp+ and Lpp E. coli K12
isolates
are nearly identical, differing in the deletion of lpp (the gene encoding MLP)
and mutations
in aroD6 (the gene encoding a 26 kDa protein) in the Lpp+ isolate, and pps-6
(the gene
encoding an 84 kDa protein) in the Lpp isolate. Pittard J and Wallace BJ,
JBacteriol
91:1494-1500 (1966); Hirota Y et al., Proc Natl Acad Sci USA 74:1417-1420
(1977). The
unmutated gene products of aroD and pps-6 have molecular weights that are
considerably
higher than that of MLP (26 and 84 kDa respectively, versus 5-9 kDa for MLP)
and are not
described to exhibit the same heterogeneity of molecular weight that is
exhibited by MLP.
Duncan K et al., Biochem J238:475-483 (1986); Geerse RH et al., Mol Gen Genet
218:348-
352 (1989). Thus it is doubtful that the difference in the pattern of staining
is due to the
mutations other than lpp.
Example 5
Identification of OMPs released by bacteria incubated in human serum
Previous studies have demonstrated that E. coli and Salmonella bacteria
incubated in
human serum release complexes of OMPs and LPS that can be affinity-purified
using O-
chain specific anti-LPS IgG. Hellman J et al., Jlnfect Dis 176:1260-1268
(1997);
Freudenberg MA et al., Microbial Pathogenesis 10:93-104 (1992). To test the
hypothesis
that OmpA, PAL, and MLP are present in OMP/LPS complexes released by bacteria
into
human serum, polyclonal anti-018 IgG was used to affinity-purify LPS from
sterile filtrates
of human serum incubated with E. coli 018K+ bacteria as described.
Methods. The following IgGs were covalently conjugated to magnetic beads
(BioMag Amine Terminated 8-4100, PerSeptive Diagnostics, Cambridge, MA)
according to
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the manufacturer's instructions and as previously described (Hellman J et al.,
Jlnfect Dis
176:1260-1268 (1997)): marine monoclonal IgG directed against the O-
polysaccharide of E.
coli 018 LPS and an unrelated marine IgG~ (ATCC, Rockville, MD), IgG from
rabbit
antisera to the E. coli 018 O-polysaccharide vaccine and to heat killed E.
coli J5, and IgG
from normal rabbit serum (normal rabbit IgG). Briefly, magnetic beads were
activated by
incubation in 5% glutaraldehyde, washed and incubated with dialyzed IgG at 5
mg IgG/ml.
The percentage of IgG covalently coupled to the beads was 85-95%. Hellman J et
al., Jlnfect
Dis 176:1260-1268 (1997).
E. coli 018K+ bacteria were grown to mid-log phase, harvested and washed. The
resultant bacterial pellet was resuspended in an equal volume of normal human
serum (10g
bacteria/ml) with ampicillin (200 ~g/ml) and incubated for 2 hours at
37°C on a rotating
drum. The serum was filtered through a 0.45 micron filter to remove intact
bacteria. The
serum filtrate was then incubated with antibody-conjugated magnetic beads.
Antibodies used
for these affinity-purification studies included: polyclonal anti-018 IgG, IgG
from J5
antiserum, and IgG from normal rabbit serum. Two hundred microliters of each
sample was
incubated with IgG-conjugated beads that had previously been washed and
resuspended in
800 microliters of PBS (final concentration of IgG 100 ~g/ml). Reaction
mixtures were
incubated for 16-20 hours at 4 °C, with end-over-end mixing. The
antibody-conjugated
beads with attached antigens were then separated from the 1:4 diluted serum by
placing the
tubes in a, strong magnetic field, and the beads were washed three times with
PBS. Antigen
was eluted by heating the beads (5 minutes, 100 °C) in 100 microliters
SDS-PAGE sample
buffer (2.5% sodium dodecylsulfate, 22% glycerol, in Tris base). Supernatants
were
carefully separated from the beads, and (3-mercaptoethanol (0.5%) and trace
bromophenol
blue were added. Twenty microliters of each sample were then electrophoresed
on lanes of
16% gels and transferred to nitrocellulose. Blots were stained with mouse anti-
J5 IgG, Mab
anti-Ol 8 IgG, and mouse monoclonal antibodies directed against each of the
OMPs (2D3,
1 C7, and 6D7). Blots were developed as described above using biotinylated
horse anti-
mouse IgG as secondary antibody.
Captured antigens were immunoblotted with the marine monoclonal IgGs against
OmpA (2D3), MLP (1C7), and PAL (6D7), Mab anti-018, and marine polyclonal anti-
J5
IgG.
Results. OmpA, PAL, and MLP were all detected in samples that were affmity-
purified using polyclonal anti-018 IgG, indicating that bacteria release
complexes containing
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these OMPs and LPS. Referring to Figure 6, eluted samples stained with marine
monoclonal
IgGs directed against OmpA (2D3), PAL (6D7), and MLP (1C7) are shown in the
three left
panels, and samples stained with polyclonal mouse anti-JS IgG and a marine
monoclonal IgG
directed to the O-polysaccharide chain of E. coli O18 LPS are shown in the two
right panels.
Samples in the various lanes correspond to those affinity-purified with:
rabbit anti-JS IgG
(lane 1), rabbit O-chain specific anti-LPS IgG (lane 2), and normal rabbit IgG
(lane 3).
Molecular weight markers are as indicated to the left of the two sets of
panels. PAL, but not
OmpA or MLP, was also detected in samples that were affinity-purified using
anti-JS IgG
(Figure 6). The OMPs were not detected in immunoblots of samples that were
affinity
purified using IgG from normal rabbit serum. The OMPs were also not detected
in
immunoblots of samples prepared from sterile filtrates of bacteria incubated
with ampicillin
without human serum.
Example 6
Model of Gram-negative sepsis in burned rats
Release of OMPs was studied in an infected burn model in rats that was adapted
from
a marine sepsis model. Stevens EJ et al., A quantitative model of invasive
Pseudomonas
infection in burn injury, JBurn Care Rehabil 15:232-235 (1994).
Methods. Male Sprague-Dawley rats weighing 225-250 gm were anesthetized with
ether (Sigma) and subjected to a 15% total body surface area full-thickness
burn by
application of heated brass bars (100 °C, 15 seconds). Rats were then
inoculated by
subcutaneous injection of E coli 018K+ (10-100 CFU) into the burned area. At
72 hours, all
rats were bacteremic and were given an intravenous dose of ceftazidime (80
mg/kg) via the
tail vein. Blood was collected into 5 mM EDTA (to prevent coagulation) by
cardiac puncture
3 hours later and diluted four-fold with PBS. Plasma was prepared by
centrifugation (200 x
g, 5 minutes, 4 °C) and then filtered (0.45 micron) to remove intact
bacteria.
Filtered plasmas from septic rats were incubated with magnetic beads
covalently
conjugated with polyclonal rabbit anti-JS IgG, antigen-nonspecific control
IgG, and anti-O-
chain specific IgG. Antibody-conjugated beads were washed and resuspended in
500
microliters of filtered rat plasma (final concentration of IgG 100~g/ml),
incubated overnight,
and washed with PBS as described above. Antigen was eluted by heating beads in
50
microliters SDS-PAGE sample buffer, and samples were further processed as
described
above. Twenty microliters of each sample containing eluted antigen was
electrophoresed on
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16% SDS-polyacrylamide gels and transferred to nitrocellulose. Captured
bacterial antigens
were assessed for the three OMPs by immunoblotting using a mixture of murine
monoclonal
antibodies (2D3, 6D7, and 1 C7) directed against each of the three OMPs as the
primary
antibody and developing by the more sensitive chemiluminescence method.
Following
transfer, nitrocellulose was blocked with 5% powdered skim milk in TTBS,
washed, and then
incubated with primary and secondary antibodies, and with avidin-biotin-
peroxidase as
described above. Blots were then rinsed three times with TTBS and developed
using equal
volumes (1-2 ml each) of enhanced luminol and oxidizing reagents (Renaissance
Chemiluminescence Reagents, NEN Lifesciences Products, Boston, MA). Film
(Reflection
Autoradiography, NEN Lifesciences Products) was exposed for 30 seconds to 1
minute.
Results. Representative data obtained from two rats are shown in the blots
presented
in Figure 7. Three bands were present in samples affinity-purified by anti-O
chain specific
IgG in 3 of 9 rats, and at least 1-2 bands were present in 6 of 9 rats. Lanes
in Figure 7
correspond to: plasma collected from bacteremic rats immediately prior to
(lane 1 ) and 3
hours after (lanes 2, 3, 4) intravenous administration of ceftazidime.
Filtered plasmas were
affinity purified with polyclonal rabbit anti-JS IgG (lanes l and 2), normal
rabbit IgG (lane
3), and polyclonal rabbit IgG directed against the O-polysaccharide side chain
of E. coli 018
LPS (lane 4). The black arrows to the right of the blots point to the 5-9 kDa,
18 kDa, and 35
kDa OMPs. The blots were developed by the more sensitive chemiluminescence
technique
which amplifies cross-reactive IgG bands (denoted by white arrows to right of
the figure).
The 18 kDa OMP was present in samples affinity purified using anti-JS IgG
(lanes 1 and 2) in
7 of 9 rats. In 2 of 9 rats there was also some capture of the 5-9 kDa and 35
kDa OMP by
anti-JS IgG.
The foregoing written specification is considered to be sufficient to enable
one
skilled in the art to practice the invention. The present invention is not to
be limited in scope
by examples provided, since the examples are intended as a single illustration
of one aspect
of the invention and other functionally equivalent embodiments are within the
scope of the
invention. Various modifications of the invention in addition to those shown
and described
herein will become apparent to those skilled in the art from the foregoing
description and fall
within the scope of the appended claims. The advantages and objects of the
invention are not
necessarily encompassed by each embodiment of the invention.
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All references, patents and patent publications that are recited in this
application are
incorporated in their entirety herein by reference.
We claim:
WO 01/13948 CA 02382221 2002-02-19 pCT~S00/22736
SEQUENCE LISTING
<110> The General Hospital Corporation
<120> Outer Membrane Protein A,
Peptidoglycan-Associated Lipoprotein, and Murein Lipoprotein
as Therapeutic Targets for Treatment of Sepsis
<130> M0765/7028W0 (AWS)
<150> US 60/149,960
<151> 1999-08-20
<160> 4
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gaggagaagc ccggttaagc ctgcggctga gttac 35
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<220>
<221> UNSURE
<222> (1)...(2)
<223> identified with reasonable confidence by mass
spectrometric sequencing
<221> UNSURE
<222> (6)...(7)
<223> cannot be unambiguously differentiated by mass
spectrometric sequencing
<221> UNSURE
WO 01/13948 cA o23a222i 2002-o2-is pCT/pS00/22736
<222> (12)...(12)
<223> cannot be unambiguously differentiated by mass
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<400> 4
Gly Val Ser Ala Asp Gln Ile Val Ser Tyr Gly Lys
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2
